BIO105 (Genetics)

Department of Biological Sciences

College of Science and Mathematics

Mindanao State University- Iligan Institute of Technology

Iligan City, Philippines

by:

Christine Cherry E. Solon, Ph.D.

Strictly for educational purposes only. Do not upload to any site nor
distribute to those who are not enrolled in this course. The images
herein are used to make understanding of the concepts easier.
Not for sale.

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REFERENCES:
Ardito, F., Giuliani, M., Perrone, D., Troiano, G., & Lo Muzio, L. (2017). The
crucial role of protein phosphorylation in cell signaling and its use as
targeted therapy (Review). International journal of molecular
medicine, 40(2), 271–280. https://doi.org/10.3892/ijmm.2017.3036

Balakrishnan, L., & Bambara, R. A. (2013). Okazaki fragment metabolism.

Cold Spring Harbor perspectives in biology, 5(2), a010173.
https://doi.org/10.1101/cshperspect.a010173

Dai, H., Liu, J., Malkas, L. H., & Hickey, R. J. (2009). Characterization of
RNA primers synthesized by the human breast cancer cell DNA
synthesome. Journal of cellular biochemistry, 106(5), 798–811.
https://doi.org/10.1002/jcb.22015

Das-Bradoo, S. & Bielinsky, A. (2010) DNA Replication and Checkpoint

Control in S Phase. Nature Education 3(9):50.

Di Huang, Xiaowei Li, Ming Sun, Tengxun Zhang, Huitang Pan, Tangren
Cheng, Jia Wang and Qixiang Zhang. (2016). Identification and
Characterization of CYC-Like Genes in Regulation of Ray Floret
Development in Chrysanthemum morifolium. Front. Plant Sci., 07
November 2016 | https://doi.org/10.3389/fpls.2016.01633

Griswold, A. (2008) Genome packaging in prokaryotes: the circular

chromosome of E. coli. Nature Education 1(1):57.

Jiang, H., Zhang, X., Chen, X., Aramsangtienchai, P., Tong, Z., & Lin, H.
(2018). Protein Lipidation: Occurrence, Mechanisms, Biological
Functions, and Enabling Technologies. Chemical reviews, 118(3),
919–988. https://doi.org/10.1021/acs.chemrev.6b00750

Klug, W.S., M. R. Cummings, C. A. Spencer and M. A. Palladino. (2012).

Concepts of Genetics. 10th ed. Pearson Education, Inc.

Kuzminov A. (2014). The precarious prokaryotic chromosome. Journal of

bacteriology, 196(10), 1793–1806. https://doi.org/10.1128/JB.00022-
14

O'Connor, C. (2008). Karyotyping for Chromosomal Abnormalities. Nature

Education. 1(1):27. Retrieved from
https://www.nature.com/scitable/topicpage/ karyotyping-for-
chromosomal-abnormalities-298/

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Pray, L. (2008). Semi-conservative DNA replication: Meselson and Stahl.
Nature Education 1(1):98

Pray, L. (2008). Major molecular events of DNA replication. Nature

Education 1(1):99.

Russell, P.J. (2010). iGenetics A Molecular Approach. 3 rd edition.

Pearson Education, Inc.

Shapiro, R.S. and L.E. Cowen. (2012). Thermal Control of Microbial

Development and Virulence: Molecular Mechanisms of Microbial
Temperature Sensing. mBio Oct 2012, 3 (5) e00238-12; DOI:
10.1128/mBio.00238-12

Verma, P.S. and V.K. Agarwal. (2004). Cell Biology, Genetics, Molecular
Biology, Evolution and Ecology. Multicolour Edition. S. Chand &
Company, Ltd.

https://www.nature.com/scitable/topicpage/chromosomes-14121320/
https://openoregon.pressbooks.pub/mhccmajorsbio/chapter/dna-
replication-in-prokaryotes/

USING THIS MODULE

Read carefully this section. It provides you an overview of the
overall approach used for the development and implementation of
learning and teaching activities.

All sessions are self-directed, which requires you to take full control of the
learning process. You need to take initiative, with or without the
assistance of your teacher, to complete all learning and assessment
activities. You have to develop and sustain your motivation to succeed in
this course.

To become successful in your learning, you need to do the following:

1. Read the learning outcomes for each session. They articulate the
knowledge you need to acquire and skills to develop.
2. Assess your prior knowledge by identifying which learning outcomes
you already know, and which learning outcomes you need to focus
on.

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 3. Develop your learning goals based on the results in #2. Keep those
learning outcomes in mind while you engage in the learning
activities.
4. After completing all learning activities, reflect which learning
outcomes/ learning goals you have achieved by accomplishing the
ROYL. A software will be utilized to check for plagiarism so copying
and pasting is prohibited. At the end of the ROYL (or activity with
questions), always include a list of refences. Copied-pasted answers
will not be credited. Answer directly to the point. Long answers do
not necessarily mean more points.

Housekeeping Rules:
1. You need to study all reading materials and complete all activities.
2. Be mindful of the schedule of activities. If circumstance/s will not
allow you to take the quiz/exam, inform your teacher within 24 hrs
through email or through private message in FB.
3. For any inquiry/clarification related to the topics, post the question
in Google Classroom. There may be changes regarding this. Just
wait for announcements from your teacher. For concerns which are
not related to the course topics but which you feel can affect your
performance, send a message through e-mail or whatever option is
given by your teacher. Questions will be answered right away if they
are asked during the agreed real-time online consultation. If
questions are asked at other times, answers might be delayed but
you will really get answers. Inquiries done during the weekend will
be answered on Monday.
4. Extension for submission would only be allowed if the reason is valid.
What is valid? Power interruption, internet connectivity problem,
real emergencies.
5. Each topic will be good for a certain duration and that will serve as
your guide in managing your schedule so that you can finish
everything within the allowable time. Schedule is posted in google
classroom or in MOLE.
6. The files for submission can be downloaded from google classroom
or MOLE and answers will be entered therein. Example: Completion
of a table where the format is already given in the module. Be sure

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 to use the proper file name, as instructed. Observe proper etiquette
in the submission of output through e-mail. Outputs should be
submitted to your teacher via e-mail. Follow basic e-mail etiquette
and include this basic information:
Subject: filename like APK3, ROYL3 or Ex7
Write a short message just to save your teachers time in replying
since automatic options are only given if there is a message in the e-
mail. Everybody is expected to GET THIS RIGHT this time.
And MAKE SURE that you really ATTACH the file that you are
submitting. If you will not get acknowledgment of your submission,
it is your duty to check or follow-up from your teacher if your
submission was really received.
7. Follow all instructions given above. Comprehension is part of your
grade for communication

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 MODULE 3: Molecular Genetics

INTRODUCTION
In this module, we will be looking at the DNA, its structure and various
conformations, how it is made, its role in directing the synthesis of RNA
and polypeptide products, regulation of gene expression at many different
levels including epigenetic regulation and mutations. This is going to be
building up so it is important that you pay attention as you study the early
parts because that will surely help you a lot when you go through the more
complex topics.

LEARNING OUTCOMES:
At the end of this module, you should be able to:
1. explain how DNA encodes genetic information.
2. explain the role of complementary base pairing in the precise
replication process of DNA.
3. describe the different forms of DNA repair and explain why DNA
repair is critical for cells.
4. discuss different components of prokaryotic and eukaryotic gene
regulation.
5. discuss different components and types of epigenetic gene
regulation.
6. define mutation and describe why cells have so many systems
devoted to avoiding or correcting mutations.
7. describe the effects of different point mutations.
8. explain why and how mutations that occur outside of coding regions of
structural genes can influence gene expression.
9. compare and contrast different mutation mechanisms.
10. relate variations in chromosome number and structure to phenotypic
variation.
11. explain the basic principles in biotechnology and infer on the pros
and cons of genetically modifying all living things.
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Activating Prior Knowledge (Individual)
Reflect on the eleven learning outcomes above. Always do this
before starting each module. This should be submitted on the first
day assigned to study each module. This is only based on what
you can remember (stored knowledge) so this should be easy to complete.
To complete the Table below, use the downloadable file (in document
format) that is posted in our google or MOLE classroom. Name and save
the file using this format: your family name (section) – APK3. E-mail the
completed document to your professor.

Any questions/clarifications
Learning
What do you know? in relation to learning
Outcomes
outcomes
1
2
3
4
5
6
7
8
9
10
11

Before we proceed to the meat of this topic, it is but proper to review

on what you know about cell structure and function. Reflect on the
following questions:
1. How can something so small as our DNA store so much information?
2. Why is there a need to make compact chromosomes out of our linear
DNA?

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3. Is there an advantage to having many replication origins as
compared to having only 1? Does it make the process of replication
faster?
4. How do prokaryotes delineate the processes of replication,
transcription and translation from each other when there is no
nucleus (with nuclear envelope) that compartmentalize the inside of
the cell?
5. Do mutations always hurt the organism? Or are they good for the
species?

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Reading Material
Our knowledge about chromosome behavior is a prelude of gene
behavior and in this module, we will be looking at the
chromosomes and genes in the molecular level.

A. The concept of the gene

In the previous module, we have enumerated some definitions of gene and
that it represents a portion of DNA that codes for a product: a protein or
any functional unit. The genes needed for the success of prokaryotes are
organized in a circular DNA in the prokaryotic cell, but not enclosed by a
nuclear envelope. In eukaryotes, the DNA is contained in the nucleus of
each cell and are linear. (There are also DNA that are contained in other
organelles like mitochondria and plastids.) Since DNA is long, it is
composed of so many genes, spanning certain portions and coding for
many different products. Remember that the products can be genes
which code for proteins and other products that perform essential
functions.

B. Molecular Structure of DNA

This is not the first time for you to learn about DNA. In the cell cycle, there
is already a mention of DNA, whose synthesis takes place during the S
phase of interphase.

DNA or deoxyribonucleic acid is a macromolecule composed of a chain of

nucleotides. Each nucleotide (fig. 1) is composed of a pentose sugar
called deoxyribose (fig. 2), which has a phosphate group attached to its
4th carbon, and a nitrogenous base attached to its 1st carbon. Adjacent
nucleotides are joined by phosphodiester bond (fig. 3), which forms
through condensation accompanied by the release of water. This is what
happens during the polymerization process in the assembly of nucleotides
into nucleic acids, and in this case, specifically, DNA is formed.

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 Fig. 1. Components
of nucleotides.

Fig. 2. The sugars in

nucleic acids. Take
note how deoxyribose
differs from ribose.

Fig. 3. Condensation reaction

during the formation of
phosphodiester bond
between adjacent
nucleotides.

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The two DNA strands are antiparallel (fig. 4). Note that the upper end of
the left strand is labelled 5’ end while the right strand is labelled 3’ end.
On the lower end of the DNA, you can find the 3’ end of the left strand and
the 5’ end of the strand at the right.

Together, sugar and phosphate form the backbone of the DNA molecule.
The nitrogenous bases which occupy the interior part of the double helix
are of 2 types: purines, which include adenine (A) and guanine (G), and
pyrimidines, which include cytosine (C) and thymine (T). Purines have
double ring structures while pyrimidines have single ring structures.
Complementary base-pairing involves hydrogen bond formation between
a purine and a pyrimidine. The pairing is specific between adenine (A) and
thymine (T) and between guanine (G) and cytosine (C).

Fig. 4. The antiparallel strands of DNA as proven by the

positioning of the sugars and also designated by
the labels at each end.

The base-pairing which involves purine and pyrimidine follows a strict

pattern, which ensures that the 2 strands are equidistant from each other
(fig. 5). If base-pairing only involves 2 purines, both being of double ring
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structures, it could be so crowded in the middle of the helix. On the other
hand, having 2 pyrimidines pair will leave too much space between them.
Having a large purine and a small pyrimidine thus satisfies the spatial
requirements of the base pairs. In addition, A–C and G–T pairs do not fit
well together; that is, they do not easily form hydrogen bonds.

Fig. 5. Distance between DNA

backbones is maintained
all throughout the DNA
molecule because of the
strict purine-pyrimidine
base pairing.

Take note of the number of H

bonds between A and T and
between G and C (fig. 6). Although
H bonds are weak bonds, so many
of them in the entire DNA molecule
can give the DNA molecule a
stable structure.

Fig. 6. Hydrogen bonds form

between complementary
base pairs.

One big contribution from Erwin Chargaff is what is known as the

Chargaff’s Rule which zeroes in on the fact that by virtue of the existing

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specific base-pairing, one can deduce that, in a given DNA molecule, there
should be the same number of adenine and thymine and the same guanine
and cytosine. Although it sounds simple now, this was a big help to
Watson and Crick when they were developing their base pair model for the
double helix structure of DNA.

This is the DNA that we know now. It is composed of

2 anti-parallel strands wound around each other in a
double helix (fig. 7).

Fig. 7. The DNA double helix which looks like a twisted

ladder or a spiral staircase.

C. Organization of DNA in prokaryotic and eukaryotic chromosomes

Prokaryotic Chromosomes
Prokaryotic cells usually have a single chromosome and one or a few
plasmids (fig. 8), which are extrachromosomal DNA molecules with their
own replicons that carry non-essential genes such as those that increase
adaptation of their host cells to specific environments or those that aid
growth in specific conditions or encode antibiotic resistance.

Fig. 8. Prokaryotic cell containing

a single DNA and plasmids.

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Prokaryotic chromosomes are almost always circular. They are either
completely devoid of centromeres or carry the so-called “plasmid
centromeres” which are not essential. In terms of condensation and
packing, prokaryotic DNA appears naked in that the isolated nucleoids
(fig. 9) look like a collection of wire loops, loosely held together by a
proteinaceous core. Prokaryotic nucleoids are always known to
segregate continuously, as they replicate, and without additional
condensation.

Fig. 9. The nucleoid

organization of
the prokaryotic
DNA.

Supercoiling (fig. 10), which is one way that prokaryotes compress their
DNA into smaller spaces, is made possible with the help of unique
topoisomerases. Supercoils are either negative or right-handed or
positive or left-handed supercoils
(fig. 11). Mesophilic prokaryotes
use DNA gyrase to create negative
supercoils while thermophilic
prokaryotes use reverse gyrase to
create positive supercoils.

Fig. 10. Supercoiling of prokaryotic

DNA.
Genomes can be negatively supercoiled, meaning that the DNA is twisted
in the opposite direction of the double helix, or positively supercoiled,
meaning that the DNA is twisted in the same direction as the double helix.
Most bacterial genomes are negatively supercoiled during normal growth.
Once the genome has been condensed, DNA
topoisomerase I, DNA gyrase, and other proteins
help maintain the supercoils.

Fig. 11. Negative versus positive supercoiling.

(modified from Shapiro and Cowen, 2012)
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Prokaryotic genomes are small but terrible. They are jam-packed with
genes with noncoding sequences accounting for an average of 12% of the
prokaryotic genome. Prokaryotic genome evolution is dominated by
horizontal gene transfer enabling them to move into any environment
compatible with the general metabolism. The organization of prokaryotic
genes into operons is often attributed to frequent horizontal gene transfer.
They have a few active mobile elements, which are always tightly
controlled, as element jumping or repeat-induced recombination in the
gene-packed prokaryotic genomes always reduces adaptation and is
often lethal.
Prokaryotic genomes strongly prefer to delete rather than insert DNA.
Such space-saving capability, indeed!

Eukaryotic Chromosome

Eukaryotic cells usually have multiple chromosomes per karyotype

(complete chromosome set), with a typical diploid number of between 10
and 100. Chromosomes are always linear and are always equipped with
centromeres (either single or multiple ones) — places of attachment of the
kinetochore proteins which are important components of the segregation
spindle.

Multiple chromosomes are better than a single chromosome as the gene

storage option, and linear chromosomes are obviously better than the
circular ones, because they avoid the potentially lethal problems of
chromosome dimerization and catenation.
Having protein-mediated long-lasting sister-chromatid cohesion and
allocating at least one centromere per chromosome, guarantees faithful
segregation during cell division.

DNA condensation and packing

Eukaryotic DNA is wrapped around protein nucleosomes and is further
organized by histones and other proteins into a coil of 30-nm solenoid
fiber.
Chromatin packing also offers an additional mechanism for controlling
gene expression. Specifically, cells can control access to their DNA by
modifying the structure of their chromatin. Highly compacted chromatin
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simply isn't accessible to the enzymes involved in DNA transcription,
replication, or repair. Thus, regions of chromatin where active
transcription is taking place (called euchromatin) are less condensed than
regions where transcription is inactive or is being actively inhibited or
repressed (called heterochromatin) (fig. 12).

Fig. 12. Packing of chromatin distinguishes euchromatin from

heterochromatin.

The differences between prokaryotic and eukaryotic chromosomes are

summarized in table 1.

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Table 1: Prokaryotic versus Eukaryotic Chromosomes.

Prokaryotic Chromosomes Eukaryotic Chromosomes

• single circular chromosome • multiple linear chromosomes

in many
• condensed in the nucleoid • condensed in a membrane-
via DNA supercoiling and the bound nucleus via histones.
binding of various
architectural proteins.
• Most prokaryotes contain • Most eukaryotes contain two
only one copy of each gene copies of each gene (i.e., they
(i.e., they are haploid). are diploid).
• Nonessential prokaryotic • Some eukaryotic genomes
genes are commonly are organized into operons,
encoded on extra- but most are not.
chromosomal plasmids.
• Extrachromosomal plasmids
are not commonly present in
eukaryotes.
• Prokaryotic genomes are • Eukaryotes contain large
efficient and compact, amounts of noncoding and
containing little repetitive repetitive DNA.
DNA.

D. Replication and Error Corrections in Replication

Mode of Replication
There are 3 modes of replication as shown in figure 13. In the semi-
conservative model, which fits our DNA’s mode of replication, after one
round of replication (2nd row), every new DNA double helix would be a
hybrid that consists of one strand of old DNA (pink) bound to one strand of
newly synthesized DNA (blue). Then, during the second round of
replication, the hybrids would separate, and each strand would pair with
a newly synthesized strand. Afterward, only half of the new DNA double
helices would be hybrids (3rd row: pink&blue); the other half would be
completely new (3rd row: blue&blue). Every subsequent round of

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replication therefore would result in fewer hybrids and more completely
new double helices.
According to the conservative model, after one round of replication, half
of the new DNA double helices would be composed of completely old, or
original, DNA (pink&pink), and the other half would be completely new
(blue&blue). Then, during the second round of replication, each double
helix would be copied in its entirety. Afterward, one-quarter of the double
helices would be completely old (3rd row: pink&pink), and three-quarters
would be completely new (3rd row: blue&blue). Thus, each subsequent
round of replication would result in a greater proportion of completely new
DNA double helices, while the number of completely original DNA double
helices would remain constant.

According to the dispersive model, every round of replication would result

in hybrids, or DNA double helices that are made up of partly original DNA
and partly new DNA (strands are partly pink and partly blue). Each
subsequent round of replication would then produce double helices with
greater amounts of new DNA (3rd row: pink segments are farther from
each other).

Fig. 13. Three modes of replication of DNA.

Prokaryotic chromosomes, with their unique replication origin (R.O.)

which is a hundred nucleotides in length (fig. 14), have a defined zone,
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called the terminus, where converging replication forks fuse. The R.O.
initiates a single replication bubble per chromosome. They can have
several replication rounds in the same chromosome, reaching a maximum
of 8:1 origin/terminus ratio (replicated DNA to unreplicated DNA). All
replication origins in the same cell always fire at once (synchronously).
Repetitive sequences in the chromosome play roles in DNA folding, DNA
replication and gene expression.

Fig. 14. The replication origin of a prokaryotic chromosome.

DNA denaturation: the melting of double-stranded DNA to generate two

single strands. This involves breakage of H bonds.
Replication bubble: the region formed after the separation of the 2 DNA
strands. In fig. 14, it is the opened-up area in the circular
chromosome on the left.
Replication fork: the Y-shaped region on each side of the replication
bubble (each arrow points to an RF in fig. 14)

Eukaryotic Replication or Synthesis of DNA

Eukaryotic chromosomes have multiple and alternative replication origins

(fig. 15), generating up to hundreds of replication bubbles per
chromosome. There are a few preferred origins that tend to fire every
replication round, but most origins fire in only a fraction of replication
rounds, and if replication is behind schedule in a particular chromosomal
region, “ad hoc” origins fire in the region, accelerating local replication.
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Termination zones where converging replication forks meet are not
defined in the eukaryotic chromosomes, since replication origin usage
differs among replication rounds, shifting the location of replication fork
fusion.
Eukaryotic chromosomes always undergo a single replication round at a
time, so that during the S phase, the ratio of maximally replicated DNA to
unreplicated DNA is always 2:1 (in contrast with prokaryotic 8:1 ratio).
Eukaryotic replication origins fire during the whole S phase, starting from
“early” to “late” S phase.
Chromosomes are segregated once their replication is complete, all at
once (ensemble segregation), after additional condensation by the mitotic
spindle and pulled toward the opposite cell poles by microtubules
attached to their centromeres.

Fig. 15. Replication origins in eukaryotic chromosome.

Why is it important for eukaryotes to have many replication origins?

• eukaryotes have more DNA to replicate
• eukaryotic DNA polymerase is slower than prokaryotic DNA pol

Rate of DNA synthesis:

Prokaryotes: 1000 nucleotides/sec
Eukaryotes: 50 nucleotides/sec (so if there is a single RO, what
would normally take 7 days will take 20 days to
complete)

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STEPS in DNA Replication

1. Unwinding of the double strands by helicase (fig. 16).

• A replication fork (RF) is thus created.

• Necessary because DNA Polymerase III uses only single-strand DNA

(ssDNA) as template. (Note: We are using the names of the
prokaryotic polymerases here.)

• This separation is maintained by the following enzymes (fig. 16 & 17):

➢ SSB (single -strand binding) proteins (Helix Destabilizing
Proteins or HDP) – keep 2 strands separated and prevents
reformation of double helix
➢ DNA helicase – binds to ssDNA near replication fork and then
moves into neighboring double-strand (ds) region forcing strands
separation
➢ DNA topoisomerase/gyrase - breaks and re-joins the DNA
double helix to relieve supercoils (twisting forced by the opening
of the helix)

Fig. 16. Unwinding of the DNA double helix.

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 Fig. 17 The enzymes involved in the unwinding of DNA helix.

2. Start of synthesis of complementary strand by DNA polymerase III

Direction of replication:
DNA Polymerase III: read nucleotide sequence on DNA template in 3’ –
5’ direction (or synthesizes new strand in 5’ – 3’ direction)
• LEADING STRAND: toward replication form in 5’ – 3’ dir.
• LAGGING STRAND: away from RF in 5’ – 3’ dir.

In fig. 18, the parental DNA strands are the red ones. The strand that
serves as the template of leading strand synthesis is the leading strand
template. It is the topmost strand. The bottommost strand is called
lagging strand template. The templates contain the sequence of
nucleotides that dictates the sequence of nucleotides that will be
assembled during the synthesis of the new strands. And that is possible
through the base-pairing property of DNA.

Fig. 18. Direction of replication of the leading and lagging strands.

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The synthesis of the leading and lagging strands occur in opposite
directions. As soon as the unwinding process has opened up the 3’ end of
the DNA template, synthesis of the leading strand starts (fig. 19), with the
assembly of a short RNA primer using ribonucleotides. This provides a
free 3’-OH for the attachment of deoxyribonucleotides during elongation
process. (RNA primers are synthesized by an enzyme called primase ,
which is a form of RNA polymerase, a close relative of DNA polymerase.)
Without this free 3’-OH on the last nucleotide of the RNA primer, the DNA
polymerase cannot start polymerization. The choice of nucleotides that
are assembled will depend on the sequence of nucleotides on the parental
(or old) DNA strand which now acts as template for the synthesis of new
(leading) strand. The assembly of nucleotides will proceed continuously
towards the replication fork extending to the 5’ end of the template strand.

Fig. 19. Synthesis of the leading strand starts when the 3’ end of the DNA
is freed from its complementary strand and continues towards the
end of the entire parental DNA.

There is a delay in the start of the lagging strand synthesis because DNA
polymerase III can only assemble nucleotides from 5’ to 3’ and not only
that, it still has to satisfy another structural requirement and that is to
maintain the anti-parallel positioning of the two DNA strands. So, it takes
some time to allow a portion of the parental DNA to free up a long-enough
part that can now serve as template for the assembly of nucleotides. But
this delay shouldn’t be taken as an indication that the whole parental DNA
molecule should unwind before synthesis of lagging strand can start. The
delay will highlight the important role of SSB proteins in making sure that
the unwound portion of the strand will remain open and the nucleotides
therein are available for base-pairing.

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The assembly of the lagging strand starts with the assembly of an RNA
primer (7-10 ribonucleotide long) a certain distance away from the 5’ end
of the parental DNA (fig. 20). This process is made possible by the enzyme
called primase. The start of lagging strand synthesis thus needs RNA
primer & primase.

Fig. 20. Start of lagging strand synthesis.

Once the primer has been assembled, DNA polymerase III starts its work
(fig. 21). It catalyzes the assembly of nucleotides based on the exposed
nucleotides on the lagging strand template (parental DNA strand).
Polymerization continues up to the end of the template strand (fig. 21-22).
The finished short segment is called an Okazaki fragment. Okazaki
fragments are ∼1200 nt (nucledotides) long in bacteria but only about 200
nt long in eukaryotes.

The first fragment extends from the start of the RNA primer which is the 5’
end to the 3’ end (which is aligned with the 5’ end of the lagging strand
template). DNA helicase has already opened up more of the parental DNA
and thus it is possible to start synthesizing another Okazaki fragment. It
occurs in a 5’-3’ direction, going away from the replication fork. Again, it
begins with an RNA primer (same process; primase is involved) followed
by joining of nucleotides by DNA polymerase III which continues until the
last nucleotide which will be positioned right beside the first nucleotide of
the RNA primer of the first Okazaki fragment (fig. 22-23).

Fig. 21. Synthesis of the lagging strand is done discontinuously.

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Imagine that the synthesis of the lagging strand will continue towards the
other end of the DNA molecule. A number of Okazaki fragments are
expected to be formed. The fragments are ultimately joined together.
Before actual joining of fragments, the ribonucleotides of the RNA primer
are removed by the enzyme DNA polymerase I. This removal will not leave
that area of the DNA blank because DNA polymerase then assembles the
replacement deoxyribonucleotides. (This also happens in the synthesis of
the leading strand. The RNA primer’s ribonucleotides will be replaced
with deoxyribonucleotides by DNA polymerase (fig. 22).

Why is there a need to use RNA primers when they are only going to be
removed and replaced later? The reason was mentioned earlier. DNA
polymerase cannot assemble nucleotides without an existing free 3’-OH
end. That can be provided by the last nucleotide of the RNA primer. (And
remember that RNA primer is synthesized by another enzyme, the
primase.)

Fig. 22. Removal of RNA primer and replacement of deoxyribonucleotides

by DNA polymerase I.

The gaps between adjacent Okazaki fragments still need to be resolved.

Joining the last nucleotide of an Okazaki fragment to the first nucleotide
of the adjacent one is done by DNA ligase which catalyzes the formation
of the phosphodiester bonds (fig. 23).

Fig. 23. Joining of Okazaki fragments by DNA ligase marks the completion
of the lagging strand synthesis.

BIO105 Module 3 25 | P a g e
To summarize the process of replication:
1. DNA helicase breaks H bonds between 2 DNA strands at the replication
origin and then continues heading to replication fork.
2. SSB proteins keep the strands separate and keep them from rewinding.
3. DNA topoisomerase relieves tension (prevents over-winding) ahead of
the replication fork by creating transient single-strand breaks (also
double-strand breaks, if necessary). This will make the work of DNA
helicase simpler and uninterrupted.
5. Primase assembles RNA primer at the end of the DNA leading strand
template through complementary base pairing.
6. DNA polymerase III assembles deoxyribonucleotides and synthesis of
the leading strand proceeds continuously.
7. On the lagging strand, RNA primer synthesized by primase starts the
synthesis of each Okazaki fragment, which marks the beginning of
lagging strand synthesis. Synthesis of lagging strand is catalyzed by
DNA polymerase III and proceeds in opposite direction, away from the
replication fork but still satisfies the 5’-3’ direction requirement.
8. This 5’-3’ direction requirement of nucleotide assembly is the reason
why there are many Okazaki fragments that are made. Since each
fragment begins with an RNA primer, later, they have to be removed by
DNA polymerase I and replaced with deoxyribonucleotides (again
through base-pairing).
9. The spaces or nicks between adjacent Okazaki fragments are joined
together through formation of phosphodiester bonds by the enzyme,
DNA ligase. And finally, the lagging strand synthesis is completed.
The entire process of DNA replication can actually be summarized into 3
major processes: Initiation, elongation and termination.
Initiation refers to the part when the 2 DNA strands separate at the
replication origin (with DNA helicase and SSB proteins at work!).
Next is elongation which starts with RNA primer synthesis followed by
polymerization by DNA polymerase by adding nucleotides to the primer
and this continues to the other end of the DNA where termination occurs.
The other end could also be the meeting points of 2 replication forks.
Termination involves the removal of the last primer sequence from the
lagging strand. After this is proofreading to check for errors.

BIO105 Module 3 26 | P a g e
To better understand the entire process, watch the videos included in our
Google or MOLE classroom. It is good to appreciate this process because
this is how all the information of what makes us who we are, survive for
several generations. Remember that if there is no DNA replication, there
is no cell division, and if a cell stops dividing, it is considered dead.
For easier recall, here are some of the major enzymes involved in DNA
replication (figs. 24-26):

Fig. 24. DNA helicase, the unzipping enzyme.

Fig. 25. Topoisomerase relieves tension at

the replication fork.

BIO105 Module 3 27 | P a g e
Fig. 26. DNA ligase joins
adjacent Okazaki
fragments.

Table 2 summarizes the differences between prokaryotic and eukaryotic

DNA replication.

Table 2. Comparison of some features of Prokaryotic and Eukaryotic DNA

Replication.

Prokaryotes Eukaryotes
Number of
5 (I, II, III, IV, IV) 5 ( , , , , )
Polymerases
I: synthesis, proofreading, : polymerization
repair, RNA primer
removal
: repair
II: repair
Functions : mitochondrial DNA
III: main enzyme involved
synthesis
in polymerization
: polymerization
IV and V: repair
: unknown function
Exonuclease
Yes Some only
activity
R.O. 1 Several
Length of
Okazaki 1000-2000 nt 150-200 nt
fragments
BIO105 Module 3 28 | P a g e
Remember that in the presentation of the DNA replication process earlier
we only mentioned the prokaryotic DNA polymerases. That was for easier
recall. While prokaryotes have the DNA polymerase III and I as
polymerizing enzymes, eukaryotes have DNA polymerase  and .

DNA is polymorphic and dynamic.

DNA can assume many conformations depending on the:
• condition of the environment,
• activity of the part involved and
• need of the organism.
These are the 3 forms of DNA (fig. 28):

Fig. 28. The different conformations of DNA.

BIO105 Module 3 29 | P a g e
The various forms of DNA differ in certain aspects are summarized in
Table 3.

Table 3. Comparison of the 3 forms of DNA.

DNA
B-DNA A-DNA Z-DNA
feature
Helix handedness Right Right Left
Base pairs (bp) per
helical turn 10.5 (~10) ~11 ~12

Vertical rise per bp (Å) 3.4 2.56 3.7

Rotation per bp () +36 +33 -30
Helical diameter (Å) 20 23 18
Major groove Wide, deep Narrow, deep Flat
Minor groove Narrow, deep Wide, shallow Narrow, deep

B-DNA (fig. 29)

• the canonical right-handed DNA helix that is the most common form
of DNA. It is the standard form being considered the conformation
adopted by nearly all sequences within a genome.
• It is a double helix made of two antiparallel strands that are held
together via hydrogen bonding in the A•T and G•C base pairs. The two
strands of the duplex are antiparallel and plectonemically coiled.
• with 10.5 bp every helical turn
• DNA exists as a cylinder of 20 Å in diameter with two grooves, a
highly accessible major and a narrower minor groove, spiraling
around the cylinder.
• the distance between the bases (rise) is 3.4 Å.
• has a smooth backbone
• conformation is actually highly variable and malleable. It can adopt
multiple conformations in response to the environment which can
affect protein recognition.

BIO105 Module 3 30 | P a g e
Fig. 29. B-DNA.

A-DNA (fig. 30)

• right-handed antiparallel helical duplex
• underwound structure that is more compact along the helix axis and
broader overall across the helix relative to B-DNA.
• with 11 base pairs per helical turn
• base pairs are tilted to about 20° with respect to the helical axis
• the base pairs are shifted to the helix periphery which creates a 9 Å
hole in the helix center
• shallow, wide minor groove and a channel associated with a deep,
narrow major groove.
• the average rise is 2.55
• has been observed under conditions of reduced water content, such
as in DNA fibers at 75% relative humidity or in solutions containing
organic solvents or high salt concentrations.
• biological relevance of A-DNA: underwinding for replication fidelity
▪ by inducing the A-form, the polymerase exploits the structural
features of the highly accessible minor-groove to ensure that
the correct base has been added relative to the template
sequence.
• an A-like DNA conformation may either form upon binding of certain
proteins to DNA, or be an important intermediate step in forming the
strongly distorted DNA conformation observed within at least some
complexes with proteins.
BIO105 Module 3 31 | P a g e
 Fig. 30. A-DNA.

Z-DNA (fig. 31)

• left-handed, the most underwound form of the double-helix, has been

mostly found in alternating purine-pyrimidine sequences (CG)n and
(TG)n. (results to zigzag backbone)
• 12 bp per helical turn, with bases shifted to the periphery of the helix
• the repeating unit is 2 bp (dinucleotide, not single base pair)
• thinner (18 Å)
• only one deep, narrow groove equivalent to the minor groove in B-
DNA.
• the major groove in Z-DNA is not so much a groove but more a convex
outer surface, while the minor groove becomes a deep, narrow and
largely inaccessible crevice.
• an average rise is 3.7 Å/bp
• the backbone follows a zigzag path (some phosphate groups are
closer and electrostatic repulsion between them is greater than in B-
DNA)
• stabilized by high salt concentrations or polyvalent cations that
shield interphosphate repulsion better than monovalent cations.
• other factors also contribute to Z-DNA stability:
If an alternating purine-pyrimidine sequence occurs in a circular
DNA molecule, DNA supercoiling is a major driving force for Z-DNA
formation. The junctions between the B- and Z-DNA in supercoiled
DNA span several base pairs in which nucleobases behave as if they
were unpaired.
• usually in locations near the site of transcription initiation.

BIO105 Module 3 32 | P a g e
 Fig. 31. Z-DNA.
The biological function of Z-DNA:

• Being the most underwound form of the double-helix, it consequently

serves as a sink for the torsional tension (superhelical tension) in
negatively supercoiled DNA. This expands the range of cellular
situations that could support the formation, at least transiently, of Z-
DNA.

• Z-DNA helps to maintain the gene (close to it) in its activated,

nucleosome-free state (nucleosomes do not bind to the very rigid Z-
DNA form) - regulation of gene expression

• Z-forming sequences accumulate near the transcription start site of

genes in humans and other eukaryotes (~80% of the genes in human
chromosome 22 have at least one Z-DNA sequence in the vicinity of
their transcription start sites)

• suggested additional functions: RNA editing and gene

transactivation.

• has several potential functions may be either beneficial or

deleterious to the cell.

BIO105 Module 3 33 | P a g e
Why do different forms of DNA exist?
There is simply not enough room for the DNA to be stretched out in a
perfect, linear B-DNA conformation. In nearly all cells, from simple
bacteria through complex eukaryotes, the DNA must be compacted by
more than a thousand-fold in order even to fit inside the cell or nucleus.
The DNA then assumes the other conformations as the need arises.

Transition of B-DNA to A-DNA

As mentioned earlier, DNA is polymorphic and dynamic which means that
in the cell, DNA can exist in any of these forms and can shift from one to
another depending on the need (activity-wise and the type of environment
the DNA is exposed to).

Fig. 32 shows how B-DNA can shift to the A-DNA conformation. Since it is
known that the B conformation is the most stable, in this case, the shift
makes the DNA less stable.

Fig. 32. How B-DNA shifts to A-DNA conformation.

Transition of B-DNA to Z-DNA

In the same way, under certain conditions already mentioned under the
characteristics of Z-DNA, B-DNA can also assume a Z conformation (fig.
33).
BIO105 Module 3 34 | P a g e
 Fig. 33. How B-DNA shifts to a Z-DNA conformation.

While we know that the B conformation is the most stable form of DNA in a
double-strand state, it is amazing that DNA can even exist as 3 or 4-
stranded structures, at least transiently (fig. 34).

Fig. 34. The 3-stranded and 4-stranded DNA structures.

What holds one strand Which DNA double helix do

against the other in you think would be harder to
the double helix? separate into two strands:
DNA composed predominantly
of AT base pairs, or of GC
base pairs? Why?
BIO105 Module 3 35 | P a g e
H-DNA: 3 strands (fig. 35)
• formed when a single DNA strand invades the
major groove of a DNA duplex (but in order for the
duplex to accommodate this third strand, it must
unwind to broaden the major groove)
• triple-stranded helices are favored in negatively
supercoiled DNA (The invading third strand can
be intermolecular or intramolecular.)
• the interaction between strands involves the
Hoogsteen (the meaning of H) edge of the Watson-
Crick base pairs of the duplex to form base
triplets which explains its name.
- Its presence in large numbers suggests that
naturally occurring DNA sequences can
cause increased mutagenesis when they
assume the non-standard DNA structure
formation Fig. 35. H-DNA.

• H-DNA is formed primarily in mirror repeat sequences (sequences

that have dyad symmetry within a strand, as in
…AGAGGGnnnGGGAGA. (If you read it backward, the sequence of
bases is the same as when you read it forward.) Mirror-repeats
occur more frequently in eukaryotic genomes and have been
documented to have effects on gene expression of several disease
related genes.
• As with Z-DNA, the repeating sequence motif of H-DNA appears to
be a source of genetic instability resulting from double-strand
breaks.

There are several conformations of DNA that can be assembled from four
strands. The three structures discussed here show very different and
unique helical forms, starting with a conformation that is most similar to
standard B-DNA, and leading through forms that differ dramatically from
the original Watson-Crick model.

BIO105 Module 3 36 | P a g e
 HJ (Holliday Junction) (fig. 36)
• essential to several cellular processes
including:
➢ recombination-dependent DNA lesion
repair
➢ viral integration
➢ restarting of stalled replication forks, and
➢ proper segregation of homologous
chromosomes during meiosis
• essential intermediates in double-strand
break repair

Fig. 36. The HJ form which is composed of 4

DNA strands.

G-quadruplexes (G refers to guanine) (fig. 37)

• The four-stranded structures assembled from guanine-rich
sequences found primarily in
➢ telomeric DNA repeats (3’-overhangs at chromosome ends)
➢ also, in central regions of the genome: like in centromeric
sequences and in the immunoglobulin switch region.

The strands are held together by pairing the Watson-

Crick edge of each guanine with the Hoogsteen edge
of an adjacent guanine, creating a cyclic
arrangement of four guanines into G-tetrads. These
tetrads are stacked with a right-handed helical twist,
and are stabilized by monovalent cations (Na + or K+)
coordinated to the O2 oxygens of the guanines, and
sandwiched between the base stacks.

Fig. 37. G-quadruplex is also composed of 4 DNA

strands.
BIO105 Module 3 37 | P a g e
 I-motifs (I refers to intercalated); also known as
I-form DNA (fig. 38) a transitory conformation
that can form in sequences rich in cytosine.
associated with C-rich sequences
Stabilized by acidic conditions, they are comprised
of two parallel-stranded DNA duplexes held together
in an antiparallel orientation by intercalated,
cytosine–cytosine base pairs.
fashioned from two parallel C-strands intercalated in
a head-to-tail fashion. The two duplexes of poly(dC)
are stabilized by base pairing the Watson-Crick
edges of two cytosines to form hemi-protonated C-
C+ pairs.

Fig. 38. I-form of DNA, with 4 strands.

Eukaryotic DNA polymerases and their functions

One of the amazing properties of DNA is its ability to correct errors and,
as much as possible, preserve the information stored in it. It is thus a good
idea to discuss on the enzymes that are involved in DNA repair, some of
which were already mentioned under DNA replication. Table 4
summarizes the major DNA polymerases in prokaryotes and eukaryotes.

Table 4. Major DNA polymerases involved in Prokaryotic and Eukaryotic

Replication.
Exonuclease
Enzyme activities Function
3’ → 5’ 5’ → 3’

Prokaryotic DNA polymerases

DNA repair, proofreading;
DNA polymerase I Yes Yes
replication
Main replicating enzyme;
DNA polymerase III Yes No
proofreading
Eukaryotic DNA polymerases
Priming during replication; no
DNA polymerase  No No
proofreading

BIO105 Module 3 38 | P a g e
Table 4. Cont’d.

Exonuclease
Enzyme activities Function
3’ → 5’ 5’ → 3’

Eukaryotic DNA polymerases

Mitochondrial DNA
DNA polymerase  Yes No
replication; proofreading
Main replicating enzyme; fill
DNA polymerase  Yes No gaps after primer removal;
proofreading
DNA polymerase  Yes No DNA repair; proofreading
DNA polymerase  No No DNA repair; no proofreading

Exonucleases are enzymes that work by cleaving nucleotides one at a

time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction
that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its
close relative is the endonuclease, which cleaves phosphodiester
bonds in the middle (endo) of a polynucleotide chain.
Eukaryotes and prokaryotes have three types of exonucleases involved in
the normal turnover of mRNA: 5’ to 3’ exonuclease, which is a
dependent decapping protein, 3’ to 5’ exonuclease, an independent
protein, and poly(A)-specific 3’ to 5’ exonuclease. (The names of the 2
exonucleases are derived from the two ends of the DNA molecule: the 5’
cap and the poly-A tail.)

Which has better proof-reading capability, the prokaryotic or

eukaryotic DNA polymerase?

Closer inspection of table 4 shows you that those with exonuclease activity
are the ones which are involved in proofreading.
To understand this exonuclease activity better, examine fig. 39 for 3’ to 5’
activity and fig. 40 for 5’ to 3’ exonuclease activity.

BIO105 Module 3 39 | P a g e
 Fig. 39. 3’→ 5’
Exonuclease
activity of DNA
polymerase.

A base-pairing error is detected (A is paired with C; so A is a wrong

nucleotide), so DNA polymerase (the yellow circle) goes back to remove it
and replace it with the correct one which is G.

Fig. 40. 5’→ 3’

Exonuclease
activity of DNA
polymerase.

The direction followed is the same as normal replication so DNA

polymerase adds the appropriate bases as it moved along the template
DNA. Since the part that the enzyme is correcting is blocked by
nucleotides, those blocking its path are removed and the enzyme
continues its job along the 5’ to 3’ direction.

BIO105 Module 3 40 | P a g e
Earlier, we have also established the importance of priming before the
activity of DNA polymerase can start. The primer provides a 3’ free -OH
end to which DNA polymerase can attach the appropriate nucleotide.
That means that without primer, there will be no DNA synthesis (fig. 41).

Fig. 41. A primer is needed before DNA

replication.

To compare the process of priming between prokaryotes to that of

eukaryotes…in prokaryotic cells, DNA primase is its own entity and works
in a complex with the DNA helicase (fig. 42). Primase, together with DNA
helicase, comprise what is called a primosome. In eukaryotic cells, DNA
primase is associated with another polymerase, DNA polymerase α, which
initiates the leading strand and all Okazaki fragments.

Fig. 42. How priming differs between prokaryotes and eukaryotes.

BIO105 Module 3 41 | P a g e
Error Correction in DNA Replication
Errors come in many forms, ranging from replication error to damage
incurred by DNA from harmful factors like chemicals, radiation and even
attack by water.
1. Replication error is an error that occurs from replication. It can be:
a. addition of a nucleotide with an incorrect base (mispairing) (fig. 43).

Fig. 43. Wrong base added

during replication.

Note that there was a mistake made in the base-pairing. A nucleotide

bearing guanine (G) was added as the pair of T. Correction involved
replacing the excised G with an A.

b. insertion or deletion (fig. 44). Insertion is when a nucleotide is added

between nucleotides and deletion is when there is a missing nucleotide
in the sequence. Both can change the reading frame of the DNA (recall
that reading is done 3 nucleotides at a time) and this affects
transcription and translation.

Fig. 44. Insertion and deletion.

BIO105 Module 3 42 | P a g e
c. Strand slippage (fig. 45). DNA strand can slip during replication and
can cause addition or deletion of a nucleotide.

Fig. 45. Slippage of one DNA strand during replication can cause either
shorten or elongate the new strand.
The newly synthesized strand is represented by the horizontal red arrow,
indicating replication is occurring in a 5’ to 3’ direction. The template
strand is in gray. In scenario 1, the newly synthesized strand loops out at
the AAAAA portion of the sequence. Because one of the A’s does not line
up with the template strand, 1 more A is added to the sequence, resulting
in a string of 6 A’s instead of just 5. This results in a new strand that has
an extra nucleotide. In scenario 2, the template strand loops out at the
TTTTT portion of the sequence. Because one of the T’s does not line up
with the newly synthesized strand, only 4 A’s are added to the new strand
instead of 5. The resulting new strand is missing a nucleotide. (There is
deletion).

2. DNA damage (fig. 46):

Chemical damage can alter DNA structure
Damage caused by radiation (example: nicks)
BIO105 Module 3 43 | P a g e
Damage due to attack by water (cause removal of an amine group from
the base group of a nucleotide or the loss of the entire base group.

abasic:
no base

Fig. 46. How the

various agents
can damage the
DNA structure.

The fidelity of a DNA polymerase is the result of accurate replication of a

desired template. So, any change caused by the damage can be
replicated if it is not corrected. More DNA damage are shown in figs. 47
and 48.

Fig. 47. More damage to DNA. Apurinic site refers to the site with a
missing purine. This is more specific than abasic.
BIO105 Module 3 44 | P a g e
Fig. 48. Various DNA damage and possible consequences of the various
DNA damage.

Repair Systems
While damage can easily affect DNA, there are however, repair systems
that operate in many ways.

1. Excision Repair System

a. Base-pair excision repair (fig. 49)
b. Nucleotide excision repair (fig. 49)
c. Short-patch and long-patch excision repair

Base Excision Repair (BER) is important for removing damaged bases that
could otherwise cause mutations by mispairing or lead to breaks in DNA
during replication. The process is initiated by DNA glycosylases, which
recognize and remove specific damaged or inappropriate bases, forming
AP sites. AP stands for apurinic/apyrimidinic (missing purine or
pyrimidine).
BIO105 Module 3 45 | P a g e
Fig. 49. DNA repair systems: (a) Base Excision Repair and (b) Nucleotide
Excision Repair.

In fig.49a, the error in the base pairing resulted from the deamination of
cytosine that caused it to become a U. Since U is not the complementary
base pair of G (U is not a natural component of DNA), the U is removed by
uracil DNA glycosylase. Thus, the site becomes an AP site and, in this
case, apyrimidinic since C is a pyrimidine. AP endonuclease then cleaves
this AP site. The resulting single-strand break can then be processed by
either short-patch (where a single nucleotide is replaced) or long-patch
BER (where 2-10 new nucleotides are synthesized). In this case, since only
1 nucleotide was removed, the short-patch repair is employed.
The replacement of the removed base is done by DNA polymerase, by
complementary base-pairing. Then DNA ligase seals the gap on each side
of the newly added nucleotide.
In fig. 49b, the damage is caused by the formation of bonds between
adjacent pyrimidines (C and T, thus labelled pyrimidine dimer). What is

BIO105 Module 3 46 | P a g e
needed in this case is nucleotide excision repair. There is an excision
nuclease that makes a cut a few nucleotides upstream (to 5’ end), thus
making the removal of a longer segment possible. The cut segment is
separated from the main DNA by DNA helicase (which breaks H bonds
between complementary base pairs). Then DNA polymerase assembles
nucleotides based on the nucleotides displayed on the surface of the
remaining DNA strand which serves as the template. Then later, the gap
between the old strand and the newly added segment is sealed by DNA
ligase.
Take note that after the removal of the damaged nucleotide, the processes
that follow are just like the regular DNA synthesis.

Short-patch excision repair varies from base-pair excision in that the

enzymes will recognize and remove "short patches" of DNA that are
damaged. These short patches of damage arise from bulky lesions such
as thymine dimers. This is radiation-induced and leads to the bond
formation between adjacent thymine bases on the same strand of DNA.
This bond leads to a distortion in the DNA that makes a short
stretch around the thymine dimer unable to base pair correctly. The short-
patch excision repair system recognizes
such distortions and cuts the damaged
strand on both sides of the damaged region
leaving a 12 base pair gap in the strand. A
helicase then unwinds the stretch of the
helix with the damage that can then be filled
and sealed with DNA polymerase and
ligase. The short-patch excision repair can
also be used to correct damage resulting
from unnatural bases (see page 46, 1st two
sentences of the first paragraph).
Long-patch excision repair follows the
same mechanism as the short-patch type
but involves a longer segment of DNA. The
difference between these 2 is shown in fig.
50.

Fig. 50. Short-patch and long-patch repair.

BIO105 Module 3 47 | P a g e
2. Mismatch repair (fig. 51)
The mismatch
repair system is
able to identify
mismatch errors
because such
damage leads to a
small distortion in
the DNA
backbone. Once it
has identified a
mismatched base
pair, it marks the
spot with a cut and
then uses an
exonuclease
to digest or "eat
up" the DNA at the
marker.
A DNA polymerase
can then fill in the
gap with the
appropriate base.

Fig. 51. Mismatch repair in prokaryotes.

Help! I’m
losing him.

BIO105 Module 3 48 | P a g e
RNA as the Genetic Material
Although DNA is considered the genetic material simply because all the
information about the organism come from it, RNA can also be partly given
that title. Considering that transcription produces RNA, it is
understandable how some could suggest that RNA can be considered
genetic material. Fig. 52 shows a comparison between DNA and RNA.

Fig. 52. Nucleic Acids.

See the differences
between DNA and
RNA.

It has been suggested that since the 2 nucleic acids are a lot alike, why
not use RNA as the genetic material instead of DNA? Let us examine the
major features of RNA first before we “decide” if that idea is feasible.

Ribonucleic Acid (RNA)

Ribonucleic acid or RNA is a nucleic acid consisting of a chain of
ribonucleotides. It is present in both prokaryotes and in eukaryotes, and
is the only genetic material of certain viruses (virus RNA). The cellular
RNA is linear and single-stranded, but in the genome of some viruses is
double-stranded.
In cellular organisms, this is the molecule that directs the intermediate
stages of protein synthesis. DNA cannot act alone, and RNA uses to
transfer this information vital for protein synthesis (production of proteins
needed by the cell for its activities and its development.) Many types of
RNA have catalytic or regulatory activity, showing much more versatile
than DNA.
BIO105 Module 3 49 | P a g e
Types of RNA

Messenger RNA (mRNA) is the type of RNA that carries information from
DNA to ribosomes, the site of protein synthesis. The nucleotide sequence
of mRNA determines the amino acid sequence of the protein. Therefore,
the mRNA is called coding RNA.
However, many RNAs do not encode proteins, and are called non-coding
RNA, originating from own genes (intron RNA) or rejected during the
process of splicing. Noncoding RNAs are transfer RNA (tRNA) and
ribosomal RNA (rRNA) that are key elements in the translation process,
and various types of RNA regulators.
Certain RNA, called ribozymes, are able to catalyze chemical reactions
such as cut and join other RNA molecules, or form peptide bonds between
amino acids.
We will re-examine these three RNA types later.

Why is DNA preferred as the genetic material?

1. It is more stable.
2. It is more easily repaired.
3. Its information is better protected (because of double strands). Double
strands allow double checking.
When DNA is replicated, the new double-stranded DNA molecule
contains one parent strand -- which serves as the template for
replication -- and one daughter strand of newly synthesized DNA. If
there is a base mismatch across the strands, as often happens after
replication, the cell can identify the correct base pair from the parent
DNA strand and repair it accordingly. For example, if at one
nucleotide position the parent strand contains a thymine and the
daughter strand a cytosine, the cell "knows" how to fix the mismatch
by following the “instructions” in the parent strand. The cell will
therefore replace the daughter strand's cytosine with an adenosine.
Since RNA is single-stranded, it cannot be repaired in this way.

BIO105 Module 3 50 | P a g e
E. Gene Function
1. Gene-Enzyme Relationship
A gene codes for a specific product, usually an enzyme, that catalyzes
certain chemical reactions in a metabolic pathway, just like that in fig. 53.

Fig. 53. The relationship of gene and enzyme

in this metabolic pathway explains
the relationship between genotype
and phenotype. A certain phenotype
is produced if the genes coding for
both enzymes are functional. If one
of those is not, then we cannot see
the expected phenotype.

In 1941, Beadle and Tatum experimented on red bread mold (fig. 54).
They bombarded the Neurospora spores with X-rays to cause mutations
in their genes. When these mutated organisms were placed in a medium
lacking in essential nutrients, most of them died because of their inability
to complete the necessary chemical reactions to survive.
However, some mutants survived, and they grew when placed in a medium
with the necessary nutrients, so Beadle and Tatum could study them.
Neurospora can use sucrose as a source for carbon, and they can use it
to complete the appropriate reactions to survive. By inducing a mutation
in the specific genes, they tested their ability to metabolize nutrients in the
medium into another form.
Beadle and Tatum then placed the Neurospora in two media. One was a
medium that they called complete, meaning it already contained many of
the components that the spores could normally synthesize. This medium
consisted of agar, inorganic salts, malt extract, yeast extract, and
glucose. From the complete medium, they were transferred to the minimal
medium, for which the organism was required to synthesize the necessary
products on its own. The minimal medium was composed of inorganic
salts, disaccharides, fats, and other complex carbon sources, as well as
biotin, the single growth factor that normal Neurospora cannot
synthesize. Their results showed that the wild-type ones can grow in
minimal medium and complete the metabolic pathway (top row, fig. 54).

BIO105 Module 3 51 | P a g e
Fig. 54. Neurospora crassa experiment by Beadle and Tatum.

Genes A, B and C are functional in wild-type molds, thus the enzyme for
each step of the metabolic pathway is functional.
In the case of class I mutants, gene A has mutated so enzyme A is not
produced. The precursor material is not converted to ornithine and the
succeeding steps of the metabolic pathway will not occur. In order for
these mutants to grow, and since genes B and C are functional, the
medium must be added with ornithine so that citrulline can be synthesized
and from it, then arginine will also be produced.
In class II mutants whose gene B has mutated, ornithine is produced from
precursor materials in the minimal medium but citrulline cannot be
produced because the needed enzyme B is absent. To make these molds
grow, citrulline must be added to the medium and since gene C is
functional, enzyme C will catalyze the conversion of citrulline to arginine.
In the case of class III mutants, arginine is added to the medium to allow
the molds to grow.

BIO105 Module 3 52 | P a g e
This, in principle, is the basis for the disorders called inborn errors of
metabolism (IEM) which usually involve defect in certain metabolic
enzymes. We will elaborate on IEMs later in this module.

2. One Gene- One enzyme Hypothesis

Each gene is responsible for directing the building of a single,
specific enzyme.

3. Protein Structure
It is important to review the structure of proteins before we proceed to the
processes that lead to their synthesis.

There are 4 levels of protein structure. Proteins are chains of amino acids.
The primary structure is the sequence of amino acids (fig. 55). The
sequence is specific for a protein, the way there is a specific arrangement
of letters to spell your name. If you change the arrangement, you may not
be able to read it.

Fig. 55. Primary structure of

protein.

In a longer chain of amino acids, it is not possible to have just a linear

structure. There is a tendency for some amino acids which are not
adjacent to interact with each other to form an alpha helix and beta
pleated sheets. These interactions are made possible by hydrogen
bonding. This is the protein’s secondary structure (fig. 56).

BIO105 Module 3 53 | P a g e
 Fig. 56. Secondary structure of
a protein.

The ensemble of formations and folds in a

single linear chain of amino acids —
sometimes called a polypeptide —
constitutes the tertiary structure of a
protein (fig. 57). This is driven many kinds
of interactions including both covalent and
non-covalent ones. The former includes
peptide and disulfide bonds and the latter
include hydrogen bonding, ionic
interactions, Van der Waals interactions,
and hydrophobic bonds.

Fig. 57. Tertiary protein structure.

Finally, the quaternary structure of a protein (fig. 58) refers to how the
multiple polypeptide chains or subunits of complex proteins associate
with each other to form a closely packed arrangement. Each of these
subunits has its own primary, secondary and tertiary structure.

BIO105 Module 3 54 | P a g e
 Fig. 58. Quaternary structure of
protein. See how the
protein subunits
associate with each other
to form a complex.

4. Collinearity of DNA and Proteins

Colinear means having corresponding parts arranged in the same linear

order. When applied to fig. 59, a gene and the protein it codes for are
collinear. The protein sequence is
determined by the arrangement of
nucleotides on DNA.

Fig. 59. The concept of collinearity as

proven by protein being
synthesized based on
instructions from DNA.

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5. The Genetic Code and its universality
The GENETIC CODE is the nucleotide base sequence on DNA which will
be translated into sequence of amino acids of a protein. Each amino acid
is represented by a triplet of bases, read in 5’ to 3’ direction.

Characteristics of the Genetic Code:

1. It is a triplet code. Each amino acid is represented by 3 nucleotides in

sequence.
2. It is non-overlapping. The mRNA is read in successive groups of 3
nucleotides.
3. It is degenerate (or redundant) since many amino acids have more than
one codon. Exceptions are in the case of methionine (start codon)
which is AUG and tryptophan which is UGG.
4. It is almost universal. There are the same codes for almost all
organisms and viruses (except in mitochondrial DNA and in certain
microbes). Nonetheless, it should be emphasized that these variances
represent only a small fraction of known cases, and that the Genetic
Code applies quite broadly, certainly to all known nuclear genes. This
is a proof that there is a common ancestor.
5. There are start and stop codons which serve as start and stop signals.
6. There is a wobble in the anticodon, referring to the fact that even if
there are 61 amino acids, there are less than 61 tRNAs because there
is a wobble in the 3rd base at the 5’ end of the anticodon, in cases when
an amino acid has more than one code and the difference is only in the
3rd base. So, a single tRNA can recognize 3 different codons at most.

The genetic code starts from the DNA sequence because all information
comes from the DNA itself. mRNA and other RNAs are made from DNA
itself. Proteins are made out of instructions from DNA (fig. 60).

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Fig. 60. Amino acid sequence is based on DNA sequence.

RECALL some major components of DNA that were mentioned under DNA
replication.
There are 2 parental DNA strands (fig. 61):
TEMPLATE strand or ANTISENSE strand: holds the information that
codes for various genes (contains anticodons)
CODING strand or SENSE strand: the complementary strand that
contains the codons.
In fig. 61, the coding strand nucleotide sequence is the same as the
sequence in the RNA strand (RED) except that for every T in the coding
strand, there is a U in the RNA strand.

a
b

Fig. 61. The parental DNA strands.

BIO105 Module 3 57 | P a g e
 The genetic code can identify the
amino acids that are assembled
to make a protein and, as
mentioned earlier, is based on the
DNA sequence. While the triplet
coding is usually used, there is
also a coding that uses single
letters to represent one amino
acid (fig. 62).

Fig. 62. The Genetic Code (Amino

acids and their one-letter
codes)

It is a lot easier to use the triplet codes. Fig. 63 is the genetic code based
on DNA sequence.

Fig. 63. Genetic code based on DNA sequence.

There are Ts instead of Us.
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 Since the amino
acids comprising a
protein are
translated using
RNA, we use the
genetic code that is
based on RNA
sequence (fig. 64).

Fig. 64. Genetic

code based on
RNA sequence.
There are Us
instead of Ts.

It is easy to remember some of these codes, like AUG that stands for
methionine, is also responsible for chain initiation. It is also good to
remember the codons that can terminate protein synthesis: UAG, UAA,
UGA. They are called chain termination codons or nonsense codons
because they do not represent any amino acid.

As can be recalled, DNA replication can be summarized into 3 steps.

Enclosed in parentheses are the major enzymes involved in each step.
1. Initiation (helicases, topoisomerases, SSB proteins, primase)
2. Elongation (DNA polymerase, ligase). Note that sometimes primase is
also considered as involved in elongation simply because it starts the
nucleotide assembly that is soon followed through by DNA polymerase.
3. Termination (DNA polymerase, telomerase, nucleases)

The entire DNA replication process takes place in the nucleus of

eukaryotes whereas it occurs in the cytoplasm in prokaryotes.
Figure 65 summarizes the continuity from DNA replication to transcription
and finally to translation. In short, proteins are made from DNA.

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Fig. 65. The Central Dogma of Genetics.

F. Transcription and Translation

Transcription
Transcription is the synthesis of RNA based on the nucleotide sequence
on a portion of DNA. It is like converting the DNA language into the RNA
language that can be subsequently used to act as instructions in making
proteins. This process takes place in the nucleus. The process is a lot like
DNA synthesis in a sense that there is also an assembly of nucleotides that
are joined together to form a chain or polymer. The sequence is based on
the sequence of nucleotides on the DNA and determined via base-pairing
properties. This time however, since RNA is the product, a different
enzyme, RNA polymerase, is involved. Since we are already familiar with
DNA polymerase, it will make it easier for us to compare RNA polymerase
with DNA polymerase. They are close relatives, anyway.

Table 5 shows how similar and different the 2 polymerases are.

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Table 5. Comparison of DNA and RNA polymerases.

DNA Polymerase RNA Polymerase

Nucleic acid
DNA RNA
synthesized

Direction of synthesis 5’ → 3’ 5’ → 3’

Template DNA (entire) DNA (portion)

dATP, dGTP, dCTP,

Substrates ATP, GTP, CTP, UTP
dTTP

Proofreading activity
Yes No
(3’ → 5’ exonuclease)

A specific DNA sequence called promoter is the binding site for RNA
polymerase or its helper proteins. RNA polymerase will open up a portion
of the DNA which is called a transcription bubble.
In prokaryotes, RNA polymerase binds directly to promoter (fig. 66).
Generally, in eukaryotes, helper proteins called general transcription
factors bind to the promoter first, helping the RNA polymerase in the cells
to identify its binding site on the DNA. This comprises the initiation step of
transcription.
Once a transcription bubble has formed, the polymerase can start
transcribing. RNA polymerase unwinds DNA by breaking H bonds. During
elongation, RNA polymerase "walks" along one strand of DNA, known as
the template strand, in the 3' to 5' direction. For each nucleotide in the
template, RNA polymerase adds a matching (complementary) RNA
nucleotide to the 3' end of the RNA strand. (So, the same with DNA
synthesis, the template is read in a 3’ to 5’ direction but the RNA is
synthesized from 5’ to 3’ end.) The RNA transcript is nearly identical to
the non-template, or coding, strand of DNA. However, RNA strands have
the base uracil (U) instead of thymine (T), as well as a slightly different
sugar in the nucleotide. So, as we can see in fig. 67, each T of the coding
strand is replaced with a U in the RNA strand.

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Fig. 66. General Information transfer: TRANSCRIPTION.

Compare the nucleotide sequence of the RNA strand (fig. 67) with the
sequence in the coding strand. For that stretch of DNA, the code is similar
in the RNA except that T in DNA are replaced with U. This proves that part
of the code in DNA is copied into the RNA.

Fig. 67. How transcription

copies part of DNA.

RNA polymerase will keep transcribing until it gets the signal to stop. The
process of ending transcription is called termination, and it happens once
the polymerase transcribes a sequence of DNA known as a terminator (fig.
68).

BIO105 Module 3 62 | P a g e
 Fig. 68. Transcription is also
composed of 3 steps:
initiation, elongation
and termination.

There are two major termination strategies found in bacteria: Rho-

dependent and Rho-independent.

In Rho-dependent termination (fig. 69), the RNA contains a binding site for
a protein called Rho factor. Rho factor binds to this sequence and starts
"climbing" up the transcript towards RNA polymerase. When it catches up
with the polymerase at the transcription bubble, Rho pulls the RNA
transcript and the template DNA strand apart, releasing the RNA molecule
and ending transcription. Another sequence found later in the DNA, called
the transcription stop point, causes RNA polymerase to pause and thus
helps Rho catch up.

Rho-independent termination (fig. 70) occurs on specific sequences in the

DNA template strand. As the RNA polymerase approaches the end of the
gene being transcribed, it hits a region rich in C and G nucleotides. The
RNA transcribed from this region folds back on itself, and the
complementary C and G nucleotides bind together. The result is a stable
hairpin that causes the polymerase to stall.

BIO105 Module 3 63 | P a g e
Fig. 69. Rho-dependent termination Fig. 70. Rho-independent
in prokaryotes. termination in
prokaryotes.

There are 3 types of RNA polymerases:

1. RNA polymerase I is located in the nucleolus and catalyzes the
synthesis of three of the RNAs found in ribosomes: the 28S, 18S, and
5.8S rRNA molecules.
2. RNA polymerase II is located in the nucleoplasm and synthesizes
messenger RNAs (mRNAs) and some small nuclear RNAs (snRNAs).
3. RNA polymerase III is located in the nucleoplasm and synthesizes:
(a) transfer RNAs (tRNAs);
(b) 5S rRNA, a small rRNA molecule found in each ribosome; and
(c) the snRNAs not made by RNA polymerase II.

All eukaryotic RNA polymerases consist of multiple subunits. Generally

prokaryotic RNA polymerases are smaller than those of eukaryotes.

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What happens to the RNA transcripts?
After termination, transcription is complete. An RNA transcript that is
ready to be used in translation is called a messenger RNA (mRNA). In
bacteria, RNA transcripts are ready to be translated right after
transcription. In fact, they're actually ready a little sooner than that:
translation may start while transcription is still going on! This is possible
because there is no true nucleus so no nuclear membrane and the DNA is
in the cytoplasm.
mRNAs are being transcribed from several different genes. Although
transcription is still in progress, ribosomes have attached each mRNA and
begun to translate it into protein. When an mRNA is being translated by
multiple ribosomes, the mRNA and ribosomes together are said to form
a polyribosome (fig. 71).

Fig. 71. Polyribosome is

made up of RNA
transcripts with
many ribosomes
attached.

In eukaryotes, the product of transcription is a pre-mRNA (or precursor

mRNA) which still undergoes modifications (fig. 72) before it is released to
the cytoplasm. Modifications include addition of a 5’ cap and a poly-A tail,
both to protect the mRNA from
being degraded by enzymes.
Non-coding sequences
(introns) are removed and
coding sequences are joined
together in the process called
splicing. The product is now
called a mature mRNA.

Fig. 72. Modifications that pre-

mRNA go through
before the mature
mRNA is released to the
cytoplasm.
BIO105 Module 3 65 | P a g e
Although not described in details here, the process involves proteins
which are regulatory in function, including activators and enhancers.
These proteins help in making the process faster and more efficient.

Types of RNA

mRNA
Messenger RNA (fig. 73), just like all the other RNAs, is synthesized using
part of the DNA as the template. This RNA molecule carries the
information that will be used in the assembly of amino acids to form a
protein. mRNAs are the transcripts of protein-coding genes.

Fig. 73. The mRNA molecule. Note the additional sequences at both ends
of the mRNA at the bottom.

tRNA
Transfer RNA (fig. 74) has a folded conformation that makes it
distinguishable from the other RNAs. It has 3 loops, with the middle loop
bearing the anticodon at its tip. Take note of the hydrogen bonds
stabilizing the looping by base-pairing. The 3’ end of the tRNA has a CCA
BIO105 Module 3 66 | P a g e
sequence to which the amino acid attaches. The tRNA is responsible for
carrying amino acids to the ribosome during protein synthesis.

Fig. 74. The many looks of the tRNA molecule.

rRNA
The ribosomal RNA (fig. 75) forms the structure of the ribosome (fig. 76)
together with ribosomal proteins. They are responsible for “reading” the
amino acid that is carried by the tRNA to the ribosomes or for translating
the information in mRNA.

Fig. 75. Ribosomal RNA (5.8S).

Fig. 76. Ribosome showing its 3 sites: E-
site (black), P-site (red) and A-
site (green).

BIO105 Module 3 67 | P a g e
Table 6 shows the differences between prokaryotic and eukaryotic
ribosomes.

Table 6. Comparison of ribosomes in prokaryotes and eukaryotes.

Large Small subunit
Type Size
subunit(rRNAs) (rRNA)
50S (5S : 120 nt, 30S
prokaryotic 70S
23S : 2906 nt) (16S : 1542 nt)
60S (5S : 121 nt,
40S
eukaryotic 80S 5.8S : 156 nt, 28S :
(18S : 1869 nt)
5070 nt)
S = svedberg unit; nt = nucleotides

Note that the S units of the subunits (or the rRNAs) cannot simply be
added. The S values are derived from the rate at which the rRNA
molecules sediment during centrifugation (sedimentation rate) and give a
very rough indication of molecular sizes. The sedimentation rate of each
subunit is affected by its shape, as well as by its mass. The nt units can be
added as these represent the integer number of units in the linear rRNA
polymers (for example, the total length of the human rRNA = 7216 nt).
Aside from the differences in their structures, different types of RNA also
perform different functions. Table 7 summarizes these.

Table 7. Roles of different kinds of RNA.

Transports amino acids to site of protein

tRNA Small
synthesis
Variable
Combines with proteins to form ribosomes; site
rRNA (several
of protein synthesis
kinds)

mRNA Variable Directs amino acid sequence of proteins

Processes initial mRNA to its mature forms (in

snRNA Small
eukaryotes)
Affects gene expression; used by scientists to
siRNA Small
knock out a gene being studied
Affects gene expression; important in growth and
MicroRNA Small
development
snRNA – small nuclear RNA; siRNA – small interfering RNA
BIO105 Module 3 68 | P a g e
TRANSLATION
One of the products of transcription is an mRNA. The mature mRNA
leaves the nucleus, goes to the cytoplasm and forms complex with the
ribosome. The entire process can be summarized into 3 steps (fig. 77)
which are the same as the steps in replication and transcription (but with
some differences in details).

Fig.77. Summary of the process of translation – 3 steps.

Translation Initiation
Initiation begins with the formation of an initiation complex. Fig. 78 is the
translation initiation process in prokaryotes.
Initiation starts with the  loading of initiation factors (IF) and binding of
GTP (guanosine triphosphate, a close relative of ATP but contains guanine
instead of adenine) to the 30s ribosomal subunit (or small subunit). Note
that there are 3 IF shown: IF1, IF2 and IF3 and they bind to specific areas
of the small ribosomal subunit.
Next is the  binding of the mRNA to the ribosome, with the part called
ribosome-binding site (or Shine-Dalgarno sequence, after the
discoverers) aligned with the mRNA-binding site of the small subunit. The
Shine-Dalgarno sequence is generally located around 8 bases upstream
of the start codon AUG. The process releases IF3 from its binding site on
the ribosome. An initiator-tRNA binds to the middle binding site of the
ribosome. This tRNA carries the amino acid methionine at one end which

BIO105 Module 3 69 | P a g e
is indicated as Met. Note that the AUG sequence on the mRNA is aligned
with the middle binding site of the ribosome. AUG is the start codon and
codes for methionine. So only a tRNA that bears a methionine can bind
there because that is what AUG specifies. The whole assembly of small
subunit, mRNA, tRNA and the GTP and IFs comprise the 30S initiation
complex.
 The large ribosomal subunit binds with the 30s initiation complex.
The
process is powered by the energy released from GTP hydrolysis. For the
first time, the ribosome is now a complete structure (70S initiation
complex) marking the
end of the initiation
process. (Recall that
the ribosomal
subunits are normally
separate when there
is no protein
synthesis.)

Fig. 78. Translation

initiation.

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Before we proceed with the rest of translation, let us look at the parts of
the ribosome (fig. 79), which we have mentioned in passing during
initiation. It is more appropriate to mention them here because at the
beginning of initiation, these sites were not yet clearly defined because
the small and large ribosomal subunits were still separate.
The fully functional ribosomes have 3 binding sites for tRNA and 1 mRNA-
binding site. The binding sites for tRNA are:
1. P-site or Peptidyl-tRNA binding site. This is the first site occupied by a
tRNA, the one called initiator tRNA. It is aligned with the AUG codon on
the mRNA.
2. A-site or Aminoacyl-tRNA binding site. This is the site which is occupied
by the incoming tRNA during
translation elongation.
3. E-site or exit site
accommodates the tRNA that
will soon be released from the
ribosome. The tRNA which
previously occupied P-site will
pass through E-site before it
totally leaves the ribosome.

Fig. 79. Parts of the ribosome.

Translation Elongation
From the descriptions given to the tRNA binding sites on the ribosome we
can also identify the specific tRNAs that would occupy those sites.
Peptidyl-tRNA is the tRNA occupying the P site while aminoacyl-tRNA is
the tRNA occupying the A site. See their “activity” during elongation (fig.
80).

BIO105 Module 3 71 | P a g e
Fig. 80. Elongation process during translation when amino acids are
joined together based on base sequence on mRNA.

Start from the topmost ribosome. Remember the order of tRNA-binding

sites: E-site on the left, P-site in the middle and A-site on the right. Notice
that only the P site is occupied. That happened during initiation.
A correct amino acid will be attached to the tRNA by an enzyme called
aminoacyl-tRNA synthetase. This process is called charging of a tRNA
meaning a tRNA that has an amino acid attached to it is a charged tRNA.
It is important to note that the specificity of codon recognition lies in the
BIO105 Module 3 72 | P a g e
tRNA molecule, not in the amino acid it carries. The tRNA thus serves as
an adapter molecule.
Elongation begins with the  binding of the aminoacyl-tRNA to the A-site.
This tRNA is escorted by EF-Tu (elongation factor thermo-unstable). The
incoming tRNA (amino-acyl tRNA) carries the amino acid coded for by the
codon displayed on the A-site. GTP hydrolysis accompanies the binding
of tRNA to the ribosome and EF-Tu is recycled with the help of EF-Ts
(Elongation factor thermo-stable) and phosphorylated (a phosphate group
is added to it), go back to the cytoplasm to later escort another incoming
tRNA.
With the 2 sites now occupied,  a peptide bond is then formed between
the amino acids attached to both tRNAs, a process that is catalyzed by the
enzyme peptidyl transferase which is present in the large ribosomal
subunit. The first amino acid gets detached from its tRNA (the one in P-
site) and it is now joined to the second amino acid that is attached to the
tRNA on the A-site by a peptide bond.
After codon-anticodon interaction, the process of  ribosome
translocation occurs. This is when the ribosome moves 3 nucleotides to
the right (3’ direction). This process is aided by EF-G. (EF-G is later on
recycled, phosphorylated and then later it will be ready for use in another
round of translocation.) As a consequence of the movement, the tRNA
from the A site is transferred to the P site (it’s now called peptidyl-tRNA)
while the one previously occupying the P site is translocated to the E site
and will soon be ejected. A-site is now open for the attachment of another
aminoacyl-tRNA. This process of translocation uses the energy stored in
GTP that is associated with EF-G (GTP hydrolysis).
Since the A-site is empty, another tRNA binds to it carrying the
appropriate amino acid that is coded by the triplet of nucleotides on the
mRNA that are now aligned at the A-site. So back to step 1 until all amino
acids coded in the mRNA are assembled.
In prokaryotes, four elongation factors are required for translation:
1. EF-Tu mediates the entry of the aminoacyl-tRNA into a free site of the
ribosome.
2. EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu,
catalyzing the release of GDP from EF-Tu.

BIO105 Module 3 73 | P a g e
3. EF-G catalyzes the translocation of the tRNA and mRNA down the
ribosome at the end of each round of polypeptide elongation.
4. EF-P stimulates peptide formation by catalyzing the first synthesis step
between the first amino acid (N-formyl-methionine) and the second
amino acid.

Translation Termination
Translation ends (fig. 81) when any of
the stop codons (fig. 82) is aligned on
the A-site. Since a stop codon does
not code for any amino acid, no tRNA
can bind to the A-site anymore. (The
last amino acid of the growing
polypeptide chain is the one that is
still joined to the tRNA which is in the
P site.) A release factor binds to the
A-site and powered by the energy
produced from the GTP hydrolysis,
the ribosomal subunits separate,
peptidyl-tRNA and mRNA are freed,
the newly formed is polypeptide is
released.

Fig. 81. Termination of translation

and the disassembly of the
translation machinery.

Fig. 82. The stop codons. The

pictures will help you
remember them easily.
UAA UAG UGA

BIO105 Module 3 74 | P a g e
If the polypeptide produced is still needed, more of it will be synthesized
using the same mRNA used earlier. If not needed, anymore, the mRNA will
be degraded and the ribonucleotides will be used for future transcription.

Despite the many similarities between prokaryotic and eukaryotic

processes, there are some obvious differences that are worth mentioning
(table 8).

Table 8. Differences between prokaryotic and eukaryotic translation:

Criteria Prokaryote Eukaryote

Larger ribosomes (more
Site Smaller ribosomes
complex rRNA and proteins)
Coupled with Separated both spatially
transcription (transcription in nucleus,
(translation can start translation in ribosome) and
Transcription-
while transcription is temporally (transcription
translation
not yet completed thus comes first and should be
polyribosomes are completed before
formed) translation is possible)
Longer (with additional 5’
Shorter
cap and poly-A tail)
mRNA Short-lived (exist for Longer-lived (can remain
some minutes then for hours before
degraded) degradation)
Shine-Dalgarno
mRNA sequence (interacts Kozak sequence (interacts
sequence with mRNA-binding site with initiator tRNA)
of ribosome)
Translation Lesser More
factors
Attached Present (guided by specific
Absent because no ER
ribosomes signal peptide)

BIO105 Module 3 75 | P a g e
Special Information Transfer
If you look at fig. 83, it is like the illustration of the central dogma but there
are elements that are added. The central dogma emphasizes on the
general information transfer. Table 9 compares how the general and
special information transfers differ. It also presents another unknown
possible mode.

Fig. 83. Modification

of the Central
Dogma diagram.

Table 9. Transfers of biological sequential information.

General Special Unknown

DNA → DNA RNA → DNA protein → DNA
DNA → RNA RNA → RNA protein → RNA
RNA → protein DNA → protein protein → protein

Reverse transcription is the transfer of information from RNA to DNA (the

reverse of normal transcription shown in fig. 83). This is known to occur in
the case of retroviruses, such as HIV, as well as in eukaryotes, in the case
of retrotransposons and telomere synthesis. It is the process by which
genetic information from RNA gets transcribed into new DNA. It is
catalyzed by reverse transcriptase.

RNA replication is the copying of one RNA to another. Many viruses

replicate this way. The enzymes that copy RNA to new RNA, called RNA-
dependent RNA polymerases, are also found in many eukaryotes where
they are involved in RNA silencing. RNA editing, in which an RNA
sequence is altered by a complex of proteins and a "guide RNA", could
also be seen as an RNA-to-RNA transfer.
BIO105 Module 3 76 | P a g e
Direct translation from DNA to protein has been demonstrated in a cell-
free system (i.e. in a test tube), using extracts from E. coli that contained
ribosomes, but not intact cells. These cell fragments could synthesize
proteins from single-stranded DNA templates isolated from other
organisms (e,g., mouse or toad), and neomycin was found to enhance this
effect. However, it was unclear whether this mechanism of translation
corresponded specifically to the genetic code.

EXERCISE 5: Molecular Basis of Inheritance

OBJECTIVES:
At the end of the exercise, you should be able to:
1. determine the base sequence on the complementary DNA strand
2. determine the RNA transcript of a specific single-stranded DNA.
3. determine the amino acid sequence corresponding to a specific
mRNA.
Procedure:
Download the worksheets from the Google/MOLE classroom.
1. You are provided with the DNA sequence in the leading strand
template. See item number 1 in worksheet, row A. Supply the
information asked for in the succeeding rows:
row B: supply the bases in the complementary strand.
row C: Assuming that the sequence in the row A is the template for
transcription, supply the bases of the mRNA transcript. Label the 5’
and 3’ ends.
row D: Identify the amino acids that comprise the polypeptide chain as
the ribosome would have identified them. Just merge cells before you
label the amino acids based on the codes. Label the 5’ and 3’ ends.
2. Complete the necessary information. Answer the guide questions.
Save your file as your family names (Section) – Ex5. E-mail completed
file to your professor following the basic e-mail guidelines.

BIO105 Module 3 77 | P a g e
G. Regulation of Gene Expression at various levels
Gene expression refers to transcription and subsequent translation of a
gene. In other words, the information that is stored in the gene is made
into a protein that will affect the phenotype of the organism. So, when we
say that a gene is turned on, it means that it is transcribed and translated
while a gene that is turned off is not. With the genome being composed of
so many genes, most of them are turned off. That can be due to functional
reasons (turned on when they are needed because their products are
needed) or it can be something due to the fact that some genes are only
needed during certain stages of development or during certain conditions
the organism’s body is in.

PROKARYOTES. Prokaryotic gene is composed of a series of gene

sequences (fig. 84).

DEFINITION OF TERMS:
OPERON: a unit of genetic function common in bacteria and phages;
consists of coordinately-regulated clusters of genes with related
functions

PROMOTER: a specific nucleotide sequence in DNA that binds RNA

polymerase and indicates where to start transcribing RNA

OPERATOR: a segment of DNA to which a transcription

factor protein bind

REGULATORY GENES: a gene involved in controlling the expression of

one or more other genes; may encode a protein, or it may work at the level
of RNA. In prokaryotes, regulator genes often code for repressor
proteins.

REPRESSOR: protein that physically obstructs the RNA polymerase from

transcribing the genes.

INHIBITOR: a gene whose presence prevents the expression of some

other gene at a different locus

INDUCER: a molecule that starts gene expression; can bind to repressors

or activators; function by disabling repressors (gene is thus expressed)

BIO105 Module 3 78 | P a g e
 Fig. 84. Prokar-
yotic gene.
Look at the
arrangement
of the genes.

Transcriptional Control Systems in Prokaryotes

These are systems that operate at the level of transcription, meaning they
can make transcription proceed or not.

Regulatory proteins can

either shut down (fig. 85) or
trigger (fig. 86)
transcription. The location
of the protein matters a lot.

Fig. 85. Negative transcriptional control.

Fig. 86. Positive transcriptional control.

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If a regulatory protein blocks the movement of the RNA polymerase, then
it controls transcription negatively. While if it is located behind the RNA
polymerase, it is stimulatory in function and can make transcription
proceed faster.

Negative Transcriptional Control Systems

Trp and Lac operons are examples of negatively controlled systems as far
as transcription is concerned.
TRP OPERON (tryptophan operon) (fig. 87): a group of genes that are
used, or transcribed, together — that codes for the components for
production of tryptophan; present in many bacteria, but was first
characterized in Escherichia coli.

Fig. 87. Parts of the trp operon. (a) The operon is on when tryptophan is
absent thus repressor is inactive. (b) The operon is off when
tryptophan is present thus activating the repressor.

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The synthesis of tryptophan is only needed when it is absent, thus, trp
operon should be turned on. Once trp is present, there is no longer a need
to synthesize it thus trp operon is turned off.
LAC OPERON (lactose operon) (fig. 88): an operon required for the
transport and metabolism of lactose in E. coli and some other enteric
bacteria; has three adjacent structural genes: lacZ, lacY, and lacA.

Fig. 88. The lac operon. (a) When lactose is absent, the repressor is active
and can bind to the operator so the operon is off. (b) In the
presence of lactose, the operon is on. It is made possible by the
binding of an allolactose, an isomer of lactose, to the repressor
which inactivates it, thus transcription can proceed.

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The lac operon contains genes that code for enzymes for lactose
metabolism so it is turned on when there is lactose. This results to the
synthesis of the enzymes -galactosidase (which catalyzes breakdown of
lactose to glucose and galactose), permease (which is involved in
transporting -galactosidase into the cell, and transacetylase (which is
involved in chemical modifications on -galactosidase). If there is no
lactose, there is no need to synthesize those enzymes, so the lac operon
is off.

Positive Transcriptional Control Systems

Positive control is also demonstrated by the lac operon. Fig. 89 shows the
effect of low glucose level on the lac operon while fig. 90 shows the effect
of high glucose level.

Fig. 89. Low glucose level

can also turn the
lac operon on.

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For the enzymes that break down lactose to be synthesized in appreciable
quantity, it is not enough that lactose be present in the bacterial cell. The
other requirement is that the simple sugar glucose must be in short
supply. Given a choice of substrates for glycolysis and other catabolic
pathways, E. coli preferentially uses glucose, the sugar most reliably
present in its environment.
How does E. coli sense the glucose concentration and how is this
information relayed to the genome? The mechanism depends on the
interaction of an allosteric regulatory protein with a small organic
molecule. The small molecule is cAMP (cyclic AMP) which accumulates
when glucose is low or absent. The regulatory protein is Catabolite
Activator Protein (CAP) and it is an activator of transcription. CAP is one
of over 300 transcription factors used by Escherichia coli alone. CAP can
promote transcription at several sites.
When cAMP binds to the allosteric site on CAP, the protein assumes its
active shape and can bind to a specific site next to the lac promoter. The
attachment of CAP to the DNA makes it easier for RNA polymerase to bind
to the adjacent promoter and start transcription of the operon. Because
CAP is a regulatory protein that directly stimulates gene expression, this
mechanism qualifies as positive regulation. The enzymes produced
catalyze breakdown of lactose to glucose and galactose. So, more
glucose is produced.
Conversely, when glucose level is high, transcription of lac operon is
turned off (fig. 90).

Fig. 90. High glucose level turns

off the lac operon.
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When there is high glucose level, cAMP level decreases and CAP is
inactive. Thus, there is no formation for cAMP-CAP complex, which causes
the RNA polymerase not to bind efficiently to mRNA and thus no
transcription takes place.

EUKARYOTES. Eukaryotic gene is long and linear. Initially it was

presented in what was called gene-battery model (fig. 91).

(a) Components of the model

(b) Redundancy of receptor sites

(c) Redundancy of integrator

genes

Fig. 91. The Britten-Davidson Model (gene-battery model). Gene-battery:

Battery – a set of structural genes controlled by 1 sensor site.

4 CLASSES OF SEQUENCES:
1. Producer genes or structural genes - Each producer may have several
receptor sites, each responding to one activator so that a single activity
though can recognize several genes. However, different activators
may activate the same gene at different times.

BIO105 Module 3 84 | P a g e
2. Receptor site or operator gene - one such receptor site is assumed to
be present adjacent to each producer gene or a set of such producer
genes
3. Integrator gene or regulator gene - responsible for synthesis of an
activator RNA that may or may not give rise to proteins before it
activates the receptor site; may also fall in cluster with same sensor
sites.
4. Sensor site – regulates activity of integrator gene which can be
transcribed only when the sensor is activated; sensor sites are
recognized by agents which, like hormones and proteins, change the
pattern of gene expression. (ex: hormone-protein complex or a
transcription factor may bind to a sensor site and cause the
transcription of integrator)

Producer and integrator genes are involved in RNA synthesis but receptor
and sensor sites help only in recognition without taking part in RNA
synthesis. Receptor sites and integrator genes may be repeated a
number of times so as to control the activity of a large number of genes in
the same cell. Repetition of receptor ensures that same activator
recognizes all of them and several enzymes of 1 pathway are
simultaneously synthesized.
As refinement to the battery model, the eukaryotic gene has the following
parts (fig. 92).

Fig. 92. Typical eukaryotic gene.

REGULATORY DNA SEQUENCES:

1. PROMOTERS (Table 10) are DNA sequences that bind to the RNA
polymerase II enzyme. They are binding sites of transcription factors.

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 Table 10. Conserved promoter elements in eukaryotes.
Conserved eukaryotic promoter Consensus sequence
elements
CAAT box GGCCAATCT
TATA box TATAA
GC box GGGCGG
CAP site TAC

See the locations of these sequences along the eukaryotic genome in fig.
92.

2. ENHANCERS are DNA sequences that, when bound by

transcription factors, enhance the transcription of an associated
gene.

Promoter, proximal elements and enhancers often are cell-type specific,

functioning only in specific differentiated cell types.

The control of eukaryotic gene expression occurs at different levels (fig.

93).

Fig. 93. Eukaryotic gene expression can be controlled at several different

steps/levels.
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Transcriptional Control
This level of control occurs during RNA synthesis. It involves, among
others, transcription factors which are proteins that control which genes
are turned on or off in the genome (fig. 94). They do so by binding to DNA
and other proteins (fig. 95 & 96).

Fig. 94. Transcription is controlled by proteins binding to

regulatory DNA sequences.

Fig. 95. The strategic looping of DNA which is caused by interaction of

various regulator proteins to the DNA at specific gene
sequences.
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 Fig. 96. See how an enhancer
associates with the
gene.

However, transcription regulators need not be located so close to the

genes that they regulate (fig. 97).

Fig. 97. Eukaryotic transcription regulators can

control gene expression even from a
distance.

The success and efficiency by which transcription regulators work is due

to the fact that they work in groups (fig. 98). That is collaboration!

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 Fig. 98. Transcription regulators work together as a “committee” to
control the expression of a eukaryotic gene.

Transcription switches allow cells to respond to changes in the

environment (fig. 99).

Fig. 99. How repressors affect gene expression.

From the previous figure, it is clear how repressors can turn genes OFF
while activators turn them ON.
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One of the major events during the start of cell division is the proper
packaging of the chromatin material into chromosomes and the purpose
is related to proper and
efficient segregation. This
also has an implication on
gene expression (fig. 100).
Genes in tightly packed
regions are off (not
expressed) while those in
loose regions are on
(expressed).

Fig. 100. Packing of

promoter DNA into
nucleosomes affects
initiation of
transcription.

A cell can change the expression of its genes in response to external

signals like hormones (fig. 101 & 102).

Fig. 101. Effect of a steroid hormone on gene

expression. Note that the hormone
enters the cell and the nucleus and
affects gene expression.
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 Fig. 102. Effect of a protein
hormone on gene
expression. Note
that the hormone
cannot enter the cell
but starts a cascade
of signal relay that
ultimately affects
gene expression.

Earlier we have seen how the lac operon is regulated by the presence or
absence of lactose. However, it also responds to glucose (fig. 103).

Fig. 103. The LAC

operon is
controlled by 2
signals.

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Post-transcriptional Control
Transcription produces immature RNA. All classes of RNA transcripts
must be processed into mature species (fig. 104).

Fig. 104. Steps in mRNA processing.

The reactions include several types and are features of both prokaryotic
and eukaryotic gene expression, and the biological consequences are
diverse.

Prokaryotic mRNA processing is relatively unimportant in regulating gene

expression. The chief function of prokaryotic mRNA seems to be to
regulate stability. The terminator stem and loop stabilize mRNA against
nucleolytic degradation, and in some cases, removal of this structure
destabilizes mRNA so that it is transcribed less efficiently.

Eukaryotic mRNA processing is much more complex and has many

consequences for gene expression. Most obviously, many eukaryotic
genes contain introns, which are found in the primary transcript. For the
mRNA to be translated into a useful protein, some way to remove them
from the transcript and still preserve the coding sequence of the mature
messenger RNA must exist.
1. 5’ capping (fig. 105): Addition of 7-methyguanylate to the 5’ end occurs
shortly after the beginning of transcription. A soluble enzyme system
BIO105 Module 3 92 | P a g e
 carries out cap addition to the 5′ end of mRNA during the time that RNA
polymerase II is still synthesizing the 3′ region of the mRNA. The 5’
methylated cap is not encoded by the DNA sequence, but is an
extension.

Shortly after transcription begins, the leading end (5’ end) is capped with
a modified guanylate nucleoside,
also called 7-methylguanylate or
m7Gpp(pN). Rather than a typical
5’ → 3’ nucleotide bond, the 5’
cap binds through a phosphate
bridge of 2 additional
phosphates, into a 5’ → 5’
configuration. This makes the
cap highly resistant to
degradation by nucleases.

Fig. 105. 5’ capping. (a) the process; (b) pre-mRNA with the 5’ cap in
place.

2. 3’ polyadenylation (fig. 106): After transcription termination, cleavage

and polyadenylation specificity factors (CPSF) bind to a poly(A) signal
sequence, typically AAUAAA, and another protein complex, cleavage
stimulation factor (CStF), interacts with a downstream G/U signal.
Binding complexes, CFI and CFII (cleavage factor I and II), help stabilize
the assembly and form a loop in the RNA. Poly(A) polymerase (PAP)
then binds and stimulates cleavage at the poly(A) site. CStF, CFI and
CFII are released and PAP adds a string of 200 -250 adenine residues
to the 3’ end.

BIO105 Module 3 93 | P a g e
 Fig. 106. Addition of
3’ poly-A tail. (a,
b) the process;
c) (c) the finished
product.

Both the 5’ cap and the 3’ poly(A) tail protect the newly synthesized mRNA
from degradation by exonucleases. Both sequences are not encoded by
the DNA template. They are extensions which are added.

3. Splicing: The eukaryotic RNA transcript has INTERRUPTED GENES

(fig. 107) which means that there are alternating introns (non-coding
sequences) and exons (coding sequences).

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Fig. 107. A representation of the premature RNA. There is alternation of
exons and introns.

Introns are the portions of the primary transcript that are removed during
RNA processing and not included in mature mRNA. It is non-coding.
Exons are the portion of the primary transcript that are retained in mature
mRNA and are translated into a protein in the cytoplasm. Exons are the
coding segments of pre-mRNA.
A typical human gene is on the order of 50,000 bp long. However, about
95% of that sequence are introns and non-coding UTRs (untranslated
regions). The average intron length in human genes is 3.3 kbp, while exons
are a mere 50 - 200 bp. In order to produce a functional mRNA, these
introns must be cut out and the exons spliced together.
Figure 108 is an estimate of the consensus sequences surrounding splice
sites based on vertebrate intron sequences. Note the G U bases at the 5’
end of the intron, the A G bases at the 3’ end and an A at the branch point
are 100% conserved in vertebrate genes. Also notice a pyrimidine rich
region, where pyrimidines occur 10 - 15 bases greater than 80% of the
time. These regions are important for the assembly of the splicing
mechanism known as the spliceosome.

Fig. 108. The pre-mRNA before splicing.

The spliceosome is made up of 5 snRNA (small nuclear RNAs) and

approximately 170 associated proteins. snRNAs are a group of small,
stable RNA molecules that are localized in the nucleus. The 5 snRNAs that
make up the spliceosome are between 107 and 220 bp in length and are
U-rich (meaning they have a predominance of the base uracil). Each of
these snRNAs associate with 6 - 10 proteins in a complex called a small
nuclear ribonucleoprotein particle or snRNP (pronounced: “snerp”).
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To remove the intron and splice the exons together, the snRNPs bind to
the 5’ end G - U sequence and the 3’ end A - G sequence (as emphasized
in fig 108). The 5’ end is then pulled towards the branch point; a splice is
made at the 5’ splice site and a phosphate bond is made between the G
and the A of the branch point. A splice is then made at the 3’ splice site
and the intron is freed from the spliceosome. A phosphate bond is then
formed between the 3’ end of
one exon and the 5’ end of the
other, thus reforming a
continuous strand of mRNA (fig.
109). The free intron, which has
formed a lariat structure, is then
degraded and the nucleotides
are recycled.
Fig. 109. Splicing. How the ends of
the adjacent exons are
joined during the removal
of the intervening intron.

To better picture this process, watch the video whose link is posted in our
Google/MOLE classroom.

So why would the cell spend so much energy producing introns that are
only to be cut out and recycled?

1. Having such large sections of non-coding DNA significantly lowers the

probability that a mutation will occur in a region that may reduce or
impair functionality.

2. It gives the cell options as to how to splice that mRNA back together
(alternative splicing) and allows the cell to produce varied gene
products with the same set of genes.

Categories of eukaryotic genes:

1. simple transcription units - those that encode a single protein
2. complex transcription units - produce a primary transcript product
that can be processed in alternate ways.

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Regulation of RNA splicing is an important part of gene
expression in higher eukaryotes.

Alternative Splicing (fig. 110): The cell can select different splice sites by
producing splicing repressor proteins or splicing activator proteins that
bind to alternate splicing sites and therefore, direct the spliceosomes to
different splicing sites or block a site altogether.

Fig. 110. Alternative splicing can produce many possible combinations of

genes that produce different proteins.

Reactions involved in RNA processing:

1. Nucleolytic cleavage – separation of mature rRNA from the primary
transcript (generated by RNA pol I)
2. Chain extension – includes synthesis/regeneration of the CCA sequence
at the 3’ end of tRNA. (For example, modified nucleotides can affect the
way in which a tRNA recognizes different codons.)
3. Nucleotide modification - example: Synthesis of methylated nucleotides
in tRNA or rRNA

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Translational Control
There are many ways by which translation is regulated:
1. The untranslated regions of mRNAs can control their
translation.
Gene expression can be controlled by regulating translation
initiation (fig. 111).

Fig. 111. There are possible changes on the mRNA that can disrupt
translation initiation.

As shown in the previous figure:

(A) Although there is base-pairing in the upstream portion of the mRNA,

the ribosome-binding site is not blocked, so protein synthesis can
start. Where there is a translation repressor protein attached to the
ribosome- binding site, no protein will be made.
(B) Sometimes the ribosome-binding site and AUG portions base pair with
upstream untranslated region and thus translation initiation is not
possible. However, it is possible to break the H bonds causing such
structural folding by using high temperature. Protein synthesis is then
possible.

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(C) Multiple loops created by base-pairing within the same mRNA
upstream from the location of the ribosome-binding site and AUG will
not affect protein synthesis. If they are included in the loop and the
loop that they are part of is cross-linked to one of the other loops, it is
not possible to even start protein synthesis.

(D) A regular mRNA without any other molecule binding to it can take part
in translation. While if an antisense RNA with the sequence
complementary with the sequence of the ribosome-binding site and
AUG binds to the mRNA at exactly the location of those 2 sequences, it
is not possible for protein synthesis to start.

2. Small regulatory RNAs control the expression of thousands of

animal and plant genes.

microRNA (miRNA): short, regulatory RNAs which control gene

expression by base-pairing with specific mRNAs and
controlling their stability and their translation

A microRNA targets a complementary mRNA transcript for destruction.

This miRNA started in the nucleus as a precursor miRNA. Once it is
completely processed and exported to the cytoplasm, it forms a complex
with RISC proteins to form the RISC (RNA-induced silencing complex) that
will search for target mRNA which contain the base sequences which are
complementary to the sequences carried by the miRNA. Those mRNAs
are identified as candidates for degradation (fig. 112).

BIO105 Module 3 99 | P a g e
 Fig. 112. How
the mRNAs
which are to
be degraded
are identified.

Post-translational Control
At the end of translation, a new protein is formed. However, there are
many different fates or destinations of these proteins so there are
modifications that ensure that they will end up where they are intended to
be. They are also important in the proper folding of the proteins. The
various post-translational processes will lead to more diversity of protein
produced. These proteins are expected to have diverse functions. So,
while the alternative splicing that can occur already causes production of
different types of proteins, the modifications after translation will
generate more of this diversity (fig. 113).

BIO105 Module 3 100 | P a g e

Fig. 113. General representation of how the genome can generate many
different types of protein products, even more than the number
of genes supposedly stored in it.

Post-translational Modifications:
Addition of chemical groups:
1. Phosphorylation: addition of a phosphate group to specific amino acid
residues on proteins (fig. 114 & 115). Protein phosphorylation is a
mechanism of regulation that is extremely important in most cellular
processes such as protein synthesis, cell cycle progression,
endocytosis, exocytosis, signal transduction, cell growth, development
and aging and apoptosis as many enzymes and receptors are activated
and deactivated via phosphorylation/dephosphorylation events due to
specific kinases (add phosphate) and phosphatases (remove
phosphate).

Fig. 114. Protein phosphor-

rylation by a kinase
and dephospho-
rylation by a
phosphatase.

BIO105 Module 3 101 | P a g e

 Fig. 115. Most commonly
phosphorylated amino
acids in mammals.

2. Glycosylation: addition of a carbohydrate, i.e. a glycosyl donor, to a

hydroxyl or other functional group of another molecule, in this case, to
a protein (fig. 116).

Fig. 116.
Glycosylation at
various groups.

Protein glycosylation, involved in cell membrane formation, is crucial to

dictate proper conformation of many membrane proteins, retain stability
on some secreted glycoproteins, and play a role in cell–cell adhesion.
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N- and O-linked glycosylation, the major forms of eukaryotic glycosylation,
have been identified in several Gram-negative and Gram-positive
bacteria.
In humans, N-linked protein glycosylation begins with the synthesis of the
oligosaccharide precursor in the cytoplasmic, which is then translocated
to the endoplasmic reticulum (ER) lumen. After the oligosaccharide
precursor undergoes several modifications, it is transferred to an
asparagine residue of a nascent protein. Some trimming of the
oligosaccharide chain is subsequently done in the ER, and the
glycoprotein moves to the Golgi apparatus, where many additional sugar
branches could be attached.
O-linked protein glycosylation starts in the Golgi apparatus with the
attachment of a GlcNAc molecule to a serine or threonine residue of a
protein. Next, multiple other sugars, such as sialic acid, can be added to
the structure.

3. Ubiquitination: the addition of ubiquitin to lysine residues of a substrate

protein. This process contains three main steps: activation,
conjugation, and ligation and requires three types of enzymes:
ubiquitin-activating
enzymes (E1s), ubiquitin-
conjugating enzymes (E2s),
and ubiquitin ligases (E3s)
(fig. 117). The system
mediates most of the
protein degradation in
eukaryotes. Someone
refers to this process as
‘the kiss of death’ because
anything that is
ubiquitinated is soon to be
degraded.
Fig. 117. Ubiquitination of a
protein that is
intended to be
degraded.
Other processes that involve ubiquitination are the cell cycle regulation,
proliferation, apoptosis, differentiation, transcriptional regulation, gene
BIO105 Module 3 103 | P a g e
expression, transcriptional regulation, signal transmission, damage
repair, autophagy and inflammatory immunity.

4. S-Nitrosylation: the addition of NO to cysteine residues of proteins (fig.

118). This is important for signal transduction such as in vascular
endothelial cells, muscle cells and some nerve endings.

Fig. 118. S-nitrosylation.

5. Methylation: the addition of methyl groups to proteins (fig. 119).

Fig. 119. Some commonly methylated amino acids.

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6. N-Terminal Acetylation: the addition of an acetyl functional group into a
protein (fig. 120). It is one of the most widespread protein modifications,
which occurs on most eukaryotic proteins, but is significantly less
common on bacterial and archaea proteins. This modification is carried
out by a family of enzymes called N-terminal acetyltransferases (NATs).
It plays an important role in the synthesis, stability and localization of
proteins. It has a considerable impact on gene expression and
metabolism.

Fig. 120. N-terminal acetylation

process.

7. Lipidation: addition of lipid moieties

to proteins (fig. 121). Lipids can be
either fatty acyl or polyisoprenyl
groups, and the modifications
typically occur on the nucleophilic
side chains of proteins. It occurs in
many proteins in eukaryotic cells
and regulates numerous biological
pathways, such as membrane
trafficking, protein secretion,
signal transduction, and apoptosis.

Fig. 121. Different types of lipids

added through lipidation.
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Each type of modification gives proteins distinct membrane affinities,
although all types of lipidation increase the hydrophobicity of a protein
and thus its affinity for membranes. The different types of lipidation are
also not mutually exclusive, in that two or more lipids can be attached to a
given protein.

Lipidation is a method to target proteins to membranes in organelles

(endoplasmic reticulum [ER], Golgi apparatus, mitochondria), vesicles
(endosomes, lysosomes) and the plasma membrane.

Types of lipidation:
C-terminal glycosyl phosphatidylinositol (GPI) anchor tethers cell surface
proteins to the plasma membrane. These are prepared in the ER, where
they are then added to the nascent protein at the same time. GPI-anchored
proteins are often localized to cholesterol- and sphingolipid-rich lipid
rafts, which act as signalling platforms on the plasma membrane.
N-terminal myristoylation gives proteins a hydrophobic handle for
membrane localization. The myristoyl group gives the protein sufficient
hydrophobicity and affinity for membranes, but not enough to permanently
anchor the protein in the membrane. It can therefore act as a
conformational localization switch, in which protein conformational
changes influence the availability of the handle for membrane attachment.
Because of this conditional localization, signal proteins that selectively
localize to membrane, are N-myristoylated.
S-palmitoylation adds a palmitoyl group from palmitoyl-CoA to the thiolate
side chain of cysteine residues via palmitoyl acyl transferases (PATs).
Because of the longer hydrophobic group, this anchor can permanently
anchor the protein to the membrane. This localization can be reversed,
though, by thioesterases that break the link between the protein and the
anchor; thus, it is used as an on/off switch to regulate membrane
localization. It is often used to strengthen other types of lipidation, such
as myristoylation or farnesylation.
S-prenylation covalently adds a farnesyl or geranylgeranyl group to
specific cysteine residues within 5 amino acids from the C-terminus via
farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II).
Unlike S-palmitoylation, S-prenylation is hydrolytically stable. It occurs in
the ER and is often part of a stepwise process of post translational
modifications that is followed by proteolytic cleavage.

BIO105 Module 3 106 | P a g e

Other types of post-translational modifications
8. Protein Cleavage (catalyzed by proteases):
Proteolytic cleavage (fig. 122 & 123) plays a central role in the
modification of protein activity, structure and localization. It also plays an
important role in several biological processes, such as in protein
degradation, digestive enzyme activation, blood coagulation, signal
transduction, rearrangement of the extracellular matrix remodelling,
polypeptide hormone development, cell invasion, metastasis, viral protein
processing, etc. Proteolytic
cleavage can occur both
inside and outside of the
cell.

Fig. 122. Processing of the

inactive proinsulin
to the active
insulin involves
the removal of the
C peptide.

Peptide bonds are indefinitely stable under physiological conditions, and

therefore cells require some mechanism to break these bonds. Proteases
comprise a family of enzymes that cleave the peptide bonds of proteins
and are critical in antigen processing, apoptosis, surface protein
shedding and cell signalling.

Fig. 123. How protein

is cut off from
the sequences
that help its
translocation.

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H. Epigenetic Gene regulation
EPIGENETICS: heritable changes in gene activity and expression (active
versus inactive genes) that do not involve changes to the underlying DNA
sequence. It is the covalent modification of DNA, protein, or RNA,
resulting in changes to the function and/or regulation of these
molecules, without altering their primary sequences.
- prefix epi- (Greek: meaning - over, outside of, around)
• includes:
1. DNA methylation
2. covalent histone modifications
3. aberrant expression of microRNAs
which all result to histone variants and chromatin remodelling.

1. DNA Methylation (fig. 124)

• the addition of a methyl group (M) to the DNA base, cytosine.
• catalyzed by DNA methyltransferase (DNMT) enzymes
• occurs in the 5’ position of the C ring, producing 5-methylcytosine
• linked to transcriptional silencing
• important for gene regulation, development and tumorigenesis

Fig. 124. DNA methylation.

Focus on the CG
with M.
Methylation of DNA is an essential epigenetic control mechanism in
mammals. It plays an important role in sex chromosome dosage
compensation, the repression of retrotransposons that threaten genome
integrity, the maintenance of genome stability, and the coordinated
expression of imprinted genes.
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Proper DNA methylation is essential for cell differentiation and embryonic
development. It has been observed to play a role in mediating gene
expression.
• Methylation near gene promoters varies considerably depending on
cell type, with more methylation of promoters correlating with low or
no transcription.
CpG islands (CGIs) are short interspersed DNA sequences (fig. 125) that
deviate significantly from the average genomic pattern by being GC-rich,
CpG-rich, and predominantly nonmethylated.

Fig. 125. CpG islands.

CpG, in genetics, is a site where cytosine (C) lies
next to guanine (G) in the DNA sequence. (The p
indicates that C and G are connected by a
phosphodiester bond.) Methylation of DNA occurs at
any CpG site.
The overall methylation levels and completeness of
methylation of particular promoters are similar in
individual humans…
• but there are significant differences in overall
and specific methylation levels among different
tissue types and between normal cells and cancer cells from the
same tissue.

Errors in methylation result to DISEASES.

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2. Covalent Histone Modifications (fig. 126)
• Involve lysine residues
• when the tails of histone molecules are acetylated at specific
locations, these molecules have less interaction with DNA, thereby
leaving it more open. Complexes of proteins called chromatin
remodelling complexes use ATP to repackage DNA in more open
configurations. Scientists have also determined that it is possible for
cells to maintain the same histone code and DNA methylation
patterns through many cell divisions. This persistence without
reliance on base pairing is called epigenetics, and there is abundant
evidence that epigenetic changes cause many human diseases.

Fig. 126. Histone

modifications. Focus
on the and .

DNA methylation and acetylation affect packaging of nucleosomes

and thus affect gene expression (fig. 127).

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 Fig. 127. Nucleosome packaging is affected by both
methylation and acetylation.

These posttranslational modifications result to the formation of histone

variants. These are critical for regulating chromatin structure and
function; they can affect transcription, recombination, DNA repair and
replication and chromosomal organization.

3. Aberrant expression of microRNAs (miRNA)

Small noncoding RNAs can also be involved in the regulatory processes
that form "silent" chromatin. MicroRNAs cause translational repression
(either at the level of initiation or elongation) or mRNA degradation (by
deadenylation) (fig. 128). They are a class of small noncoding RNAs
produced from either their own genes or introns/exons of other genes.
They bind to target mRNAs with complete or incomplete
complementarities and/or degrade/modify target mRNAs to suppress
protein translation. Therefore, one miRNA may target multiple mRNAs and
one mRNA may be regulated by different miRNAs.
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 Fig. 128. How miRNAs can regulate translation at different steps.

EPIGENETIC DISEASES
Cancer
An accumulation of genetic and epigenetic errors can transform a normal
cell into an invasive or metastatic tumor cell. Additionally, DNA
methylation patterns may cause abnormal expression of cancer-
associated genes. Global histone modification patterns are also found to
correlate with cancers such as prostate, breast, and pancreatic cancer.
Subsequently, epigenetic changes can be used as biomarkers for the
molecular diagnosis of early cancer.

Oncogenes: a gene that in certain circumstances can transform a cell into

a tumor cell.
Genes of colorectal cancer cells were substantially hypomethylated
compared with normal tissues (fig. 129).
• DNA hypomethylation can activate oncogenes and
initiate chromosome instability
• DNA hypermethylation initiates silencing of tumor suppressor genes
(TSG).

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Fig. 129. Cancer is caused by alteration of normal DNA methylation.

Imprinting
- another process involved in eukaryotic gene regulation
- involves the silencing of one of the two alleles of a gene for a cell's entire
life span.
- affects a minority of genes (include several important growth regulators)
For some genes, the maternal copy is always silenced, while for different
genes, the paternal copy is always silenced, e.g. Prader-Willi syndrome
and Angelman syndrome – two very different imprint disorders, but they
are both linked to the same imprinted region of chromosome 15. Some of
the genes in this region are silenced in the egg, and at least one gene is
silenced in the sperm. The epigenetic marks placed on these genes during
egg or sperm formation are faithfully copied into each subsequent cell,
thereby affecting these genes throughout the life of the organism.

Imprint disorders:
Prader-Willi syndrome and Angelman syndrome, both cases of mental
retardation, display an ABNORMAL PHENOTYPE as a result of the
absence (deletion) of the paternal (PWS) or maternal (AS) copy of a gene.
There is a genetic deletion in chromosome 15 in a majority of patients. The

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same gene on the corresponding chromosome cannot compensate for the
deletion because it has been turned off by methylation.
For most genes, we inherit two working copies -- one from mom and one
from dad. But with imprinted genes, we inherit only one working copy.
A role for aberrant methylation mediated by folate levels has been
suggested as a factor in Alzheimer’s disease; also, some preliminary
evidence supports a model that incorporates both genetic and epigenetic
contributions in the causation of autism. Autism has been
linked to the region on chromosome 15 that is responsible for
Prader-Willi syndrome (fig. 130) and Angelman syndrome (fig.
131). Findings at autopsy of brain tissue from patients with
autism have revealed a deficiency in MECP2 (MECP2 gene
encodes a protein that binds to methylated DNA) expression
that appears to account for reduced expression of several
relevant genes.

Fig. 130. Prader-

Willi syndrome.

Fig. 131. Angelman

syndrome.

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Immunity-Related Disorder: Lupus erythematosus (fig. 132)
Abnormal DNA methylation has been observed in patients with lupus
whose T cells exhibit decreased DNA methyltransferase (DNMT) activity
and hypomethylated DNA.
Dysregulation of this pathway leads to overexpression of methylation-
sensitive genes such as the leukocyte function-associated factor (LFA1),
which causes lupus-like autoimmunity.
LFA1 expression is also required for the development of arthritis,
which raises the possibility that altered DNA methylation patterns
may contribute to other diseases displaying idiopathic
autoimmunity. (Idiopathic - relating to or denoting any disease or
condition that arises spontaneously or for which the cause is
unknown.)

Fig. 132. Lupus erythematosus.

Take note of the
butterfly-shaped rash
on the face.

Neuropsychiatric disorders:
Schizophrenia and mood disorders are associated with:
1. DNA rearrangements - include the DNMT genes.
DNMT1 is selectively overexpressed in gamma- aminobutyric acid
(GABA)-ergic interneurons of schizophrenic brains.
2. hypermethylation has been shown to repress expression of Reelin (a
protein required for normal neurotransmission, memory formation and
synaptic plasticity) in brain tissue from patients with schizophrenia and
patients with bipolar illness and psychosis.
Synaptic plasticity is the ability of synapses to strengthen or weaken over time, in
response to increases or decreases in their activity.

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Pediatric Syndromes:
Epigenetic alterations plus specific mutations affecting components of the
epigenetic pathway have been identified.

Rett Syndrome (fig. 133)

MECP2 gene encodes a protein that binds to methylated DNA; mutations
in this protein cause abnormal gene expression patterns within the first
year of life.
Girls with Rett syndrome display reduced brain growth, loss of
developmental milestones and profound mental disabilities.

Fig. 133. Rett syndrome.

Males with mutations in the MECP2 gene often die in infancy. However, a
small number of males with a genetic change involving MECP2 have
developed signs and symptoms similar to those of Rett syndrome,
including intellectual disability, seizures, and movement problems. In
males, this condition is described as MECP2-related severe neonatal
encephalopathy.

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ATR-X syndrome (-thalassemia/mental retardation syndrome, X-linked)
(fig. 134) is characterized by severe developmental deficiencies due to
loss of ATRX, a protein involved in maintaining the condensed, inactive
state of DNA.

Fig. 134.
Children with
ATR-X syndrome.

Together, this group of clinical pediatric syndromes is associated with

alterations in genes and chromosomal regions necessary for proper
neurologic and physical development.

DNA repeats in Facioscapulohumeral Muscular Dystrophy (fig. 135)

The addition of methyl groups turns off (silences) genes, so

hypermethylated regions of DNA tend to have fewer genes that are turned
on (active). Facioscapulohumeral muscular dystrophy results when the
D4Z4 region is hypomethylated, with a shortage of attached methyl
groups.

Fig. 135. A child with Facioscapulohumeral Muscular Dystrophy.

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I. Mutation
Mutation refers to any change in DNA sequence. These changes occur at
many different levels, and they can have widely differing consequences.

Types of Mutation Based on Location (fig. 136):

1. Somatic mutations affect somatic cells so they affect only the individual
that carries them, while others affect all of the carrier
organism's offspring, and further descendants.
2. Germline (germinal) mutations occur in cells that produce the next
generation, and affect the hereditary material.

Fig. 136. Types of

mutations and
how they differ
from each
other.

Figures 137-139 are examples of somatic mutations.

Are somatic mutations ever passed on to progeny? No. It is impossible,

because somatic cells by definition are those that are never transmitted
to progeny. However, note that, if we take a plant cutting from a stem or
leaf that includes a mutant somatic sector, the plant that grows from the
cutting may develop germinal tissue out of the mutant sector. Put another
way, a branch bearing flowers can grow out of the mutant somatic sector.
Hence, what arose as a somatic mutation can be transmitted sexually.

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Fig. 137. Mutations in lung cells and in pancreatic
islet cells caused lung cancer and
diabetes, respectively.

Fig. 138. Parent dogs

(same breed) with
solid colors
produce puppy
with 2 colors.

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Example of Somatic Mutation in Plants (fig. 139):
• Flowers of new color (fig. 139a)
• Double or semi-double flowers (fig. 139b)
• Variegation (fig. 139c)
• Albinism (fig 139d)
• Colored foliage (fig. 139e)
• Fasciation (fig. 139f)
b

e
f

Fig. 139. Some somatic mutations in plants.

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1. Change in the Chromosome Structure
Chromosomal Aberrations: Changes in Chromosome Structure
• These rearrangements can occur by two mechanisms:
a. breakage and re-joining
b. crossing-over between segments of repetitive DNA.
• often due to problems that occur during meiosis or by mutagens
• can result in changes in number of chromosomes in a cell or changes
in the structure of a chromosome
Types of Chromosomal Aberrations:
• Deficiencies or deletions: loss of a chromosome segment
• Duplications or repeats: gain of a chromosome segment
• Inversions: rotation of a segment of a chromosome by 180°
• Translocations: the joining of a fragmented chromosome to a non-
homologous chromosome

DELETION (fig. 140):

Types (fig. 141):
a. terminal deletion – terminal section of chromosome is absent;
involves only 1 break on the chromosome
b. Interstitial or intercalary deletion – intermediate section of
chromosome is lost; involves 2 breaks, one on each end of
the deleted region.

Fig. 140. Deletion.

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 Fig. 141. Types of
Deletion. The
squiggle arrows
in the middle row
point to the site of
breakage on the
chromosome.

Genetic Effects of Deletion:

a. production of unique phenotypes
examples: Notch-wing mutation in Drosophila
Cri-du-chat (cry of the cat) syndrome in human babies
Turner syndrome in humans

DUPLICATION
Types:
a. tandem/repeat duplication (fig. 142) – duplicating segment gets
incorporated next to the corresponding segment; if the
segment involved and it is terminally located (and
duplicated), it is called terminal tandem. The tandem and
terminal tandem duplications in fig. 139 are of same order.
b. Reverse tandem duplication (fig.
142) - duplicating segment gets
incorporated next to the
corresponding segment but order
is reversed.

Fig. 142. Comparison of tandem

and reverse tandem
duplication.
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c. Displaced tandem duplication – the segment is repeated somewhere
away from its original location
1). homobrachial displacement – repetition of a segment on same
chromosome arm
2). heterobrachial displacement – repetition of a segment on another
chromosome arm (fig. 143)

Fig. 143. Heterobrachial type of

displaced tandem
duplication (rightmost)

d. transposition – when the segment is duplicated on the non-

homologous chromosome; fig. 144 shows the difference
between transposition and duplication within a
chromosome (intrachromosomal)

Fig. 144. Intrachromosomal transposition.

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Genetic Effects of Duplication:
a. unequal crossing over
b. production of distinct phenotypes
example: bar eye in Drosophila
c. may protect the organism from the effects of a deleterious recessive
gene or from an otherwise lethal deletion.

INVERSION involves a rotation of a part of a chromosome or a set of genes

by 180 on its own axis (fig. 145).
Types:
a. Paracentric inversion –
centromere is not included in
the inversion
b. Pericentric inversion –
centromere is included in the
inversion

Fig. 145. Types of inversion.

The location of the inverted segment can be detected cytologically in the

meiotic nuclei of such heterozygotes by the presence of an inversion loop
(fig. 146) in the paired homologs.

Fig. 146. Pericentric inversion

and the formation
inversion loop.
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Crossing over in inversion heterozygotes produces deletions,
duplications and other configurations.
Crossing over in heterozygous pericentric inversion results in deletions
and duplications and also rod-shaped (acrocentric) chromosomes. Half
of the meiotic products are non-functional and inviable. One-fourth have
normal chromosome order, one-fourth have inverted arrangement.
Crossing over in heterozygous paracentric inversion produces dicentric
chromosomes which form anaphase bridges (because they are pulled by
spindle fibers which are anchored to opposite poles). The bridge later
rupture because of the pull and fragments are produced. Deletions occur.
An acentric fragment is formed which usually fails to move to any the
poles. Half of the products are non-functional, half have normal
chromosomes, one-fourth are functional with inverted chromosome.
Thus, heterozygotes for paracentric inversions are highly sterile and
produce only parent-like progeny.

Advantages of inversions:
Fertility of inversion homozygotes and sterility of inversion heterozygotes
lead to establishment of two group (or varieties) which are mutually fertile
but do not breed well with the rest of the species. Both varieties evolve in
different directions and later become reproductively isolated species.
There is plenty of cytological evidence to prove that such evolutionary
mechanisms have and are operating in Drosophila and a number of other
organisms.

TRANSLOCATION. The piece of chromosome detaches from one

chromosome and moves to a new position on another chromosome
Types:
a. Reciprocal translocation - a single break in two homologous
chromosomes produces an exchange of chromosome segment
between them; mutual exchange (swapping) of segments between
non-homologous chromosomes (fig. 147)
b. Non-reciprocal translocation - a single break in the chromosome occurs
and it is transferred onto the end of the other; unequal exchange
between non-homologous chromosomes (fig. 147)

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c. Shift or intercalary translocation - 3 breaks, so that a two-break section
of one chromosome is inserted within the break produced in a non-
homologous chromosome (fig. 148).

Fig. 147. Comparison of non-reciprocal and reciprocal translocation.

Intrachromosomal translocation transfers the segment to another part
of the same chromosome while interchromosomal translocation
transfers the segment to another chromosome.

Fig. 148. Shift translocation.

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Translocation heterozygotes are marked by considerable degree of
meiotic irregularity. In order to affect pairing of all homologous segments,
peculiar and characteristic formations occur during synapsis. Typically, a
cross-shaped configuration is seen in prophase I.
Variations in Chromosome morphology
Isochromosomes - a chromosome in which both arms are identical. It is
thought to arise when a centromere divides in the wrong plane, yielding
two daughter chromosomes, each of which carries the information of one
arm only but present twice.
Ring chromosomes - Sometimes breaks occur at each end of the
chromosome and broken ends are joined to form a ring chromosome.
Robertsonian translocation - whole arm fusions occur in the non-
homologous chromosomes
How can translocations alter the phenotype?
• The break may occur within a gene, destroying its function.
• Translocated genes may come under the influence of different
promoters and enhancers so that their expression is altered.
• The breakpoint may occur within a gene creating a hybrid gene.
(the hybrid gene can be transcribed and translated into a protein
with an N-terminal of one normal cell protein coupled to the C-
terminal of another)
Humans normally have 23 pairs of chromosomes. Chromosome tests look
for abnormal changes within chromosomes. Such changes include parts
of a chromosome being erased, expanded, or switched. Abnormal
changes in chromosomes often occur in cancer cells. Translocation is one
of the abnormal changes sometimes found in cancer cells. Translocation
is the attachment of a piece of one chromosome to another chromosome.
The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia
chromosome, which is created by translocation. The Philadelphia
chromosome is made of parts from chromosome 9 and 22. The short
bottom piece of chromosome 9 and the short top piece of chromosome 22
attach to one another. See figure 149. This translocation creates a longer
chromosome 9 and a shorter chromosome 22. The shorter chromosome
22 is called the Philadelphia chromosome. As a result of this translation,
the ABL gene from chromosome 9 and the BCR gene from chromosome
22 join together and form the BCR-ABL fusion gene.

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 • Bone marrow cytogenetics is one type of chromosome test. It is also
called conventional cytogenetics. It is used to detect the
Philadelphia chromosome and measure the number of cells that have
it.
Fig. 149. Chromosomal
changes in Philadelphia
chromosome
characteristic of chronic
myeloid leukemia (CML)

2. Change in chromosome number

POLYPLOIDY: a chromosome mutation that
results in individuals with more than one
haploid set of chromosomes in a cell (fig.
150)

Fig. 150. Polyploidy: diploidy, triploidy and

tetraploidy.

Categories of Polyploid:

TRIPLOIDS are formed from the union of a diploid gamete and a haploid
gamete (fig. 148).

Fig. 151. How triploids

individuals are
produced.

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TETRAPLOIDS are formed from the union of 2 diploid gametes (fig. 152).

Fig. 152. How tetraploids

are produced.

Polyploidy is common in plants (fig. 153 & 154).

Fig. 153.
Polyploidy in
plants.

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Fig. 154. Polyploidy in plants.
Those with odd numbers of chromosome sets are usually sterile (fig. 155)
and so are those with odd numbers of chromosomes (fig. 156).

Fig. 155. Plant species having odd numbers of sets of chromosomes.

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Fig. 156. Some problems with some species having odd numbers of
chromosomes as in the case of the hybrid with 21
chromosomes.

Physical Characteristics of Polyploids:

a. heterosis or hybrid vigor (fig. 157)

Heterosis causes
polyploids to be more
vigorous than their
diploid progenitors.

Fig. 157. The

advantage of
heterosis.

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There is an increase in the performance of hybrids over that of purebreds
from linebred families, most noticeably in traits like fertility and sterility.
Polyploids tend to be larger than their diploid counterparts. They have
larger cells with increased
volume. They tend to display
semi-sterility or sterility. They
have higher resistance to
pests and pathogens. They
also have higher evolutionary
potential.

Fig. 158. Protection from

recessive mutations
in polyploids.

b. Polyploids are shielded from the deleterious effect of recessive

mutations (fig. 158).
Gene redundancy (result of gene duplication) shields polyploids from the
deleterious effect of mutations. There is masking of recessive alleles by
dominant wild-type alleles, an effect can act at two life stages, the first of
which is the gametophytic, haploid stage. They have the ability to diversify
gene function by altering redundant copies of important or essential
genes.

c. ability to reproduce even in the absence of sexual mates (asexual

reproduction)
Advantage: Asexual reproduction, for which the mechanistic connection
to polyploidy is unclear, enables polyploids to reproduce in the absence
of sexual mates.

d. susceptible to errors in meiosis and mitosis, usually due to spindle

irregularities

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Disadvantage: the natural tendency of polyploid mitosis and meiosis to
produce aneuploid cells
e. higher tendency for changes in gene expression
Disadvantage: the epigenetic instability that results in transgressive (non-
additive) gene regulation. (In particular, recent data on gene regulation
in polyploids provide interesting but still incomplete information on the
genetic responses that are involved in polyploidy and on the role of
epigenetic remodeling.)
- the potentially disrupting effects of nuclear and cell
enlargement

Autopolyploids versus allopolyploids:

- both have multiple sets of chromosomes
Autopolyploids (fig. 159): chromosomes are of the same type and origin
- results from mutation in
chromosome number

Fig. 159. Autopolyploids.

Allopolyploids (fig. 160): chromosomes are of different type and origin

- results from concurrent hybridization and mutations in
chromosome number

Fig. 160. Allopolyploids.

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Segregation and Linkage in Polyploids
allopolyploids: undergo bivalent pairing at meiosis (only homologous
chromosomes pair)
autopolyploids: all homologous chromosomes can pair at the same time
(equal opportunities to pair) at meiosis. Homologous chromosomes may
switch partners leading to multivalent formation (>2 chromosomes pair)
(fig. 161); double reductions (fig. 162) also formed.

Fig. 161. Multivalent formation in

polyploids.

Fig. 162. Double

reduction.

Double reduction arises from a combination of 3 major events during

meiosis:
a. crossing over between non-sister chromatids
b. an appropriated pattern of disjunction
c. the subsequent migration of the chromosomal segments carrying a pair
of sister alleles to the same gamete.

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ANEUPLOIDY: a chromosome mutation that causes individuals to have an
abnormal number of chromosomes
Aneuploid cells occur as a result of chromosome breakage or non-
disjunction errors that happen during meiosis or mitosis (fig. 163).

Fig. 163. How aneuploid cells are produced.

Nondisjunction is the failure of homologous chromosomes to separate

properly during cell division. It produces individuals with either extra or
missing chromosomes.

Common types of Aneuploidy in Humans:

Monosomy: 1 less chromosome; (23 x 2) – 1 = 45 (fig. 164)

Fig. 164. How monosomy is generated.

Trisomy: 1 additional chromosome; (23 x 2) + 1 = 47 (fig. 165)

Fig. 165. How trisomy is produced.

Examples of aneuploidy in humans:

Monosomy
• Turner syndrome (45, X) – more details under human sex anomalies
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Trisomy
• Trisomy 21 (standard) Down Syndrome (47, XX +21) or (47, XY +21)
• Trisomy 18, Edwards Syndrome
• Trisomy 13, Patau Syndrome
• Trisomy 8 Mosaicism Syndrome (T8mS)
• Klinefelter syndrome (47, XXY) – more details under human sex
anomalies
• Trisomy X (47, XXX) – more details under human sex anomalies

1. Trisomy 21 or Down Syndrome (fig. 166)

Fig. 166. Karyotype of a female with

Trisomy 21.

Trisomy 21 is one of the most common causes of human birth defects.

Symptoms vary from person to person and can range from mild to severe.

Characteristic features of humans with this trisomy are shown in figure

167.

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 Fig. 167. Characteristic features
associated with
Down Syndrome.

Persons with this condition have an increased risk of certain types of

leukemia, which can also cause early death.
The level of intellectual disability varies, but is usually moderate. Adults
with Down syndrome have an increased risk of dementia.

Types of Down Syndrome:

a. Trisomy 21 - ~95% of the cases
b. Translocation Down syndrome (~3%): occurs when an extra part or a
whole extra chromosome 21 is present, but it is attached or
“translocated” to a different chromosome rather than being a separate
chromosome 21.
3. Mosaic Down syndrome: affects about 2% of the people with Down
syndrome

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2. Trisomy 18 or Edwards Syndrome (fig. 168)

Fig. 168. Karyotype of a human

with Trisomy 18.

Characteristic features of
individuals having this
trisomy include:
congenital heart defects,
growth retardation,
dysmorphic features facial
clefts, spina bifida, severe
developmental delay.
More features in fig. 169.

Fig. 169. Additional

detailed descriptions
of babies with
Edwards syndrome.

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 Trisomy 18 types:
a. Full trisomy 18 - ~95% of the cases
b. Partial trisomy 18 (translocation type) – very rare
c. Mosaic trisomy 18 – very rare

3. Trisomy 13 or Patau Syndrome (fig. 170)

Fig. 170. (a) Karyotype of individuals

with Trisomy 13, and (b)
their characteristic
features.

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Trisomy 13 types:
a. Trisomy 13
b. Mosaicism
c. Partial trisomy 13 (translocation type)

4. Trisomy 8 Mosaicism Syndrome (T8mS) is also known as Warkany

syndrome after Dr Josef Warkany. Trisomy 8 mosaicism (T8M) is a
chromosome disorder caused by the presence of a complete extra
chromosome 8 in some cells of the body (fig. 171). The remaining cells
have the usual number of 46 chromosomes, with two copies of
chromosome 8 in each cell.

Fig. 171. Karyotype of T8mS

individual.

Characteristic features include:

• a pear-shaped, bulbous nose with upturned nostrils, a protruding
lower lip and large ears.
• stiff joints with a limited range of movement; clenched or bent fingers
and/or toes; deep palm and sole creases; occasionally under-
developed nails; missing or small kneecaps
• typically have mild to moderate intellectual disabilities.
• large ears, deep plantar furrows
• spina bifida, renal and ureteral anomalies, congenital heart disease
• increased risk of hematologic malignancy

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3. Gene mutations are alterations in the sequence of nucleotides in DNA
Common causes:
a. environmental factors - can cause base changes and even change
in DNA shape
b. mitotic/meiotic errors - can result to point mutations and frameshift
mutations

Types of Gene Mutations based on Molecular Change:

1. microlesions: base-pair substitution or point mutations (fig. 172-175)
2. frameshift mutations: caused by deletion or insertion of 1 or more
nucleotides into the original DNA sequence

Types of base-pair substitution:

Silent mutation has no effect

on amino acid sequence
meaning the base substitution
did not change the amino acid
(fig. 172).

Fig. 172. Silent mutation.

In missense mutation, the base

substitution changes the amino
acid (fig. 173).

Fig. 173. Missense mutation.

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 The base substitution that results to
nonsense mutation (fig 174) changes
the code into something that does not
code for any amino acid. A stop
codon is formed instead.

Fig. 174. Nonsense mutation.

There are also terms given to substitutions based on the type of

nitrogenous base that substituted the original base (fig. 175).

Fig. 175. Point mutations where a purine is changed for a

pyrimidine or vice versa (transversion) or a
pyrimidine is changed to another pyrimidine or
purine with another purine (transition).
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A transversion can be spontaneous, or can be caused by ionizing
radiation or alkylating agents. It can only be reversed by a
spontaneous reversion.

Frameshift mutations (fig. 176), on the other hand, cause a change in the
reading frame of the base sequence as a result of either insertion or
deletion of a base. Reading is always done 3 bases at a time so anything
missing or added can cause the shift of the reading frame.

Fig. 176. Frameshift mutations.

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Types of Mutations according to origin:
a. Spontaneous mutations – occur suddenly in nature; origin unknown
• result of normal biological or chemical processes in the organism
• often occur during the enzymatic process of DNA replication
• Mutation rate is defined as the likelihood that a gene will undergo a
mutation in a single generation or in forming a single gamete. It is
the frequency with which genes mutate spontaneously. The favored
regions of mutations are called mutation hot spots.
b. Induced mutations – artificially brought about by exposure to factors
like radiation, chemicals and extreme temperature. The inducers are
called mutagenic agents which can be natural of artificial.

MUTATION RATE is influenced by various factors:

1. genetic control: Certain genes may increase the mutation rate.
Mutator genes: genes that elevate the genomic mutation rate (or
increases spontaneous mutation rate of other genes)
• include genes that take part in DNA synthesis, such as the genes
encoding DNA polymerase; other mutator genes are involved in DNA
repair
In bacteria, these evolved to enable them to survive in fluctuating
environments. Mutator genes are found to be likely to induce
deleterious mutations and thus suffer an indirect selective
disadvantage. At the same time, bacteria carrying them can increase
in frequency only by generating beneficial mutations at other loci.
Mutator genes include genes that take part in DNA synthesis, such as
the genes encoding DNA polymerase. Other mutator genes are involved
in DNA repair. They have been analyzed in detail in bacteria, although
less is known for humans. Nonetheless, it appears that certain inherited
forms of colon cancer are due to defects in genes involved in mismatch
repair. This, in turn, increases the mutation rate of all other genes
including the tumor-suppressor genes. Defects in mutator gene are
generally recessive, like those in typical tumor-suppressor genes.

Suppressor genes: may decrease mutation rate.

In both bacteria and eukaryotes, spontaneous mutations are most
frequently caused by transposons.
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2. viral control
3. environmental control: radiation, chemicals and temperature

Mutagenic agents (fig. 177):

1) radiation
a) ionizing radiations: X-rays and gamma rays, alpha and beta
rays, electrons, protons, neutrons and other fast-moving particles
- cause breaks in polysugar phosphate backbone of DNA and,
thus, causing chromosomal mutations such as break,
deletion, addition, inversion and translocation.
b) non-ionizing radiations (ultraviolet and visible light)
- cause dimerization (thymine-thymine interactions involving
adjacent thymine) that disrupts the base-pairing with their
base pairs in the complementary DNA strand causing
distortion of double helix
2) chemicals – causes:
a) direct gene change like deamination (examples: conversion of
adenine to hypoxanthine and cytosine to uracil by nitrous
acid)
b) copy error – certain chemical compounds called base analogs
resemble certain DNA bases and can be incorporated into the
DNA in place of the real bases
3) temperature – high temperatures (increase by 10C from normal
temperature increases mutation rate by affecting thermal
stability of DNA and the rate of reaction of DNA with other
substances, most likely proteins.

The effects of these mutagenic agents on DNA are included in the topic on
DNA errors (see pages 41 and 42).

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 Fig. 177.
Mutagenic
agents.

Types of Mutations according to direction:

a. forward mutations – when wild-type phenotypes become abnormal
Forward mutations inactivate a gene.
b. reverse (back) mutations – error correcting mechanism for a forward
mutation
Types of Reverse Mutation:
1). True reversion – exact reversal of the original mutation
2). Second-site reversion – another mutation at another site in the
gene; its effects may compensate for the first mutation

Types of mutations based on Phenotypic Effects:

a. Morphological mutations: give rise to altered forms. Also called visible
mutations (affect morphological traits)
Example: those that alter normal or wild-type phenotype in Drosophila
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b. Lethal mutations: result in nonviable organisms.
Example: gene mutation in bacteria that deprives it of an enzyme. Not
providing the medium with that missing product will cause death of the
bacteria (relate this to Beadle and Tatum’s experiment on red bread
mold)
c. Conditional mutation: gives a mutant phenotype under restrictive
conditions (environmental) and causes a wild-type phenotype under
permissive conditions.
Examples: temperature-sensitive mutations in Siamese cats and
Himalayan rabbits
d. Biochemical mutations: affect the function of proteins that can affect
the well-being and survival of the affected individual; result in the
inability to carry out a specific biochemical pathway
Examples: inborn errors of metabolism
e. Neutral mutations: can occur either in a protein-coding region or in any
part of the genome, and whose effect on the genetic fitness of the
organism is negligible
Example: mutation that can change AAA (lysine) to AGA (arginine) may
have insignificant effect to function of the protein since the 2 amino
acids are chemically similar. Another thing is most of the mutations
affect introns and they are non-coding so no effect on gene products or
on gene expression.
f. Behavioural mutations: mutations that affect the behavioral patterns of
an organism
Example: the mating behavior of a fruit fly may be impaired if it cannot
beat its wings. However, the defect may be in the flight muscles, the
nerves leading to them, or the brain, where the nerve impulses that
initiate wing movements originate
f. Loss-of-function (null) mutations (LOF): reduces or eliminates the
function of the gene product. Any type of mutation, from a point
mutation to deletion of the entire gene, may lead to a loss of function.
Mostly recessive, however a dominant effect of a loss-of-function
mutation can occur during a situation known as haploinsufficiency. In
diploid organisms, haploinsufficiency occurs when the single
functional copy of the gene does not produce enough gene product to
bring about a wild-type phenotype.
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 Examples: Marfan syndrome in humans that is due to LOF mutation in
one copy of the FBN1 gene.
Also, in humans, mutations on tumor suppressor genes (TSG) or (anti-
oncogenes) such as p53 or rb, which keep the cellular processes in
check and regulate the cell cycle by repression. They result to loss of
control on the cellular proliferation, thereby allowing the cell to
progress to a cancerous state.
f. Gain-of-function mutation (GOF): results in a gene product with
enhanced or new functions. This may be due to a change in the amino
acid sequence of the protein that confers a new activity, or it may result
from a mutation in a regulatory region of the gene, leading to expression
of the gene at higher levels, or the synthesis of the gene product at
abnormal times, or places. Most gain-of-function mutations are
dominant.
Example: In humans, mutations on proto-oncogenes (genes that gain
cancerous properties when mutated) convert them into oncogenes.
Examples of proto-oncogenes are ras, myc, raf. They are regulators of
proliferation and transcription, and mutation causes them to remain in
a “switched on” state, leading to uncontrolled proliferation and
transcription, and eventually leading to a cancerous mass of cells.
g. Regulatory mutations: those that affect the regulation of gene
expression by disrupting normal regulatory processes and
inappropriately activating or inactivating expression of a gene; may
also occur in regions such as splice junctions, promoters, or other
regulatory regions of a gene that affect many aspects of gene
regulation including transcription initiation, mRNA splicing, and mRNA
stability.

mutation hot spots: DNA sequences that appear to be highly susceptible

to mutation

Mutations may contribute to cellular functional decline during aging (fig.

178).

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Fig. 178. Accumulation of mutations with age.

In a young organism, a substantial number of mutations have already

accumulated, for example, as replication errors during the very high rate
of cell division that occurs during development. These mutations may
affect the function of cells in different organs and tissues, but they are not
significant enough to affect the organism from operating optimally. During
aging, mutations accumulate further and quickly begin to exceed the
threshold for functioning optimally, which has been set low since there is
no selective advantage in maintaining genome integrity for much longer
than the age of first reproduction. Hence, there are more and more cells
that suffer functional decline and even death (open space). Occasionally,
particular combinations of mutations affect growth restraint, and such
cells grow into hyperplastic or neoplastic lesions. Functional decline is
indicated by the blue gradient, with darker shades indicating greater
deficits. Red dots and yellow triangles represent noncancer driver
mutations and cancer driver mutations, respectively.

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4. transposons
Transposons/jumping genes are also known as transposable elements
(TEs). These are DNA segments that can move around to different
positions in the genome of a single cell. Found in almost all organisms
including humans, their movement and accumulation represent a major
force shaping the genes and genomes of almost all organisms. They can
act as naturally occurring mutagens. If in their new location they insert
themselves into the coding region of a gene, they can alter the reading
frame or introduce stop codons. If they insert into the regulatory region of
a gene, they can disrupt proper gene expression. They can also create
chromosomal damage, including double-stranded breaks, inversions, and
translocations.

In the process, they may:

• cause mutations
• increase or decrease the amount of DNA in the genome of the cell (and
if the cell is the precursor of a gamete, in the genomes of any of the
descendants)
Types of eukaryotic transposons (fig. 179):
a. Class I (RNA) transposons - retrotransposons that first transcribe DNA
into RNA and then use reverse transcriptase to make a DNA copy of the
RNA to insert in a new location
• Copy–and-paste transposons: move by a "copy and paste"
mechanism but in contrast to the transposons described above, the
copy is made of RNA, not DNA. The RNA copies are then transcribed
back into DNA — using a reverse transcriptase — and these are
inserted into new locations in the genome.
Types of Class I transposons:
1) LTR (long-term repeat) retrotransposons: characterized by the
presence of long-term repeats (LTRs) on both ends (over 1000 base
pairs in each)
2) non-LTR retrotransposons: similar in structure and life cycle to
retroviruses; lack LTRs
(a) LINEs (Long interspersed elements): diverse between individual
human genomes making them useful markers for DNA
"fingerprinting".
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 (b) SINEs (Short interspersed elements): short DNA sequences
(100–400 base pairs) that represent reverse-transcribed
RNA molecules originally transcribed by RNA polymerase III; that
is, molecules of tRNA, 5S rRNA, and some other small nuclear
RNAs.
The human genome is nearly 45% TEs, the vast majority of which are
families of Class I elements called LINEs and SINEs.
b. Class II (DNA) transposons - also known as Class II TEs, these are
mobile DNA that move utilizing a single or double-stranded DNA
intermediate
• Cut-and-paste transposons: the transposon leaves one locus and
integrates at another
Some subclasses of eukaryotic DNA transposons:
1) “cut-and-paste” transposons - those that excise as double-stranded
DNA and reinsert elsewhere in the genome
2) helitrons – rolling-circle transposons
3) mavericks – self-replicating transposons

Fig. 179. Types of transposons.

Transposons are mutagens. They can cause mutations in several ways:

1. If a transposon inserts itself into a functional gene, it will probably
damage it. Insertion into exons, introns, and even into DNA flanking the
genes (which may contain promoters and enhancers) can destroy or
alter the gene's activity. The insertion of a retrotransposon in the DNA
flanking a gene for pigment synthesis is thought to have produced
white grapes from a black-skinned ancestor. Later, the loss of that
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 retrotransposon produced the red-skinned grape varieties cultivated
today.
2. Faulty repair of the gap left at the old site (in cut and paste
transposition) can lead to mutation there.
3. The presence of a string of identical repeated sequences presents a
problem for precise pairing during meiosis. Pairing can result to
unequal crossover, one of the commonest causes of duplications.

HIV, the cause of AIDS, and other human retroviruses (e.g., HTLV-1, the
human T-cell leukemia/lymphoma virus) behave like retrotransposons.
The RNA genome of HIV-1 contains a gene for reverse
transcriptase (enzyme that catalyzes reverse transcription) and one for
integrase (enzyme that catalyzes integration of viral DNA into the host
cell). The integrase serves the same function as the transposases of DNA
transposons. The DNA copies can be inserted anywhere in the genome.
Molecules of both enzymes are incorporated in the virus particle.

EXERCISE 6: Mutations
LEARNING OBJECTIVES: At the end of this exercise, you are
expected to:
1. demonstrate how gene mutations impact genetic sequences.
2. illustrate how mutations are inherited.
3. demonstrate the various types of gene mutations.
Procedure:
1. Download the worksheets from the Google or MOLE classroom.
Given the same DNA sequence as in Exercise 5, do the following:
a. deletion of the base at position 6 from 5’ end.
b. Answer the question provided.
c. Refer to the given DNA sequence in number 1 and insert a C at
position 8 from 5’ end. Place a red arrow and point it to the inserted
base.
d. Answer the question provided.
2. Answer all the questions briefly.

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Complete the necessary information. Save your file as your family names
(Section) – Ex6 and submit following the basic e-mail guidelines.

5. Practical applications and evolutionary significance of mutations

Practical Applications:
a. Improvement of lineage of crops and animals: mutating a particular
gene to improve qualities of offspring and food sources.
b. Determination of gene function: mutating a gene can reveal its function

Evolutionary Significance of Mutations:

Mutations are the raw materials of evolution. They bring about:
• formation of new alleles
• creation of new regulatory regions
Evolutionary change is based on the accumulation of many mutations with
small effects. Selection favors mutations that result in adaptive
phenotypes and eliminates non-adaptive ones. Even when mutations
produce recessive alleles that are seldom expressed in phenotypes, they
become part of a vast reservoir of hidden variability that can show up in
future generations. Such potentially recessive alleles add to the genetic
load of a population. Even mutations that have a neutral effect can
become advantageous or harmful if the environment changes to select for
or against them.

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J. Human Sex anomalies and Inborn Errors in Metabolism
HUMAN SEX ANOMALIES
1. Trisomy X (47,XXX) (fig.
180)
• incidence: 1 in 1000
female births
• above average stature
• normal phenotype
• most have learning
disabilities
• behavior problems
common
• many never diagnosed

Fig. 180. Karyotype of a

female with Trisomy X.

2. Klinefelter Syndrome (47,XXY) (fig. 181-182)

• 1:1000 male births

• tall stature
• gynecomastia
• hypogonadism
• infertility
• learning disabilities
• problems with
socialization
• many never diagnosed

Fig. 181. Karyotype of an

individual with
Klinefelter Syndrome.

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Fig. 182. Characteristic features of a person with Klinefelter Syndrome.

3. Jacob Syndrome (47, XYY) (fig. 183)

• 1/1000 newborn males
• tall stature
• most phenotypically normal
• normal IQ but 50% have learning disabilities (language and speech)
• many never diagnosed

Fig. 183. Jacob syndrome characteristic features.

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Most have normal sexual development and are able to father children.
Also possible:
• delayed development of motor skills (such as sitting and walking)
• weak muscle tone (hypotonia) hand tremors or other involuntary
movements (motor tics)
• behavioral and emotional difficulties
The rate of trisomies increases with maternal age.

5. Monosomy X (45,X) or Turner Syndrome (fig. 184-185): Turner

syndrome results when one normal X chromosome is present in a
female's cells and the other sex chromosome is missing or
structurally altered. The missing genetic material affects
development before and after birth.

Fig. 184. Turner Syndrome karyotype.

Signs and symptoms may vary significantly (probably showing prenatally

or at birth or during infancy and even during teen and adult years of a
female)
Other causes (aside from monosomy):
a. Mosaicism – due to errors in cell division during early fetal development;
chromosomal change in only some of the cells (There is chromosomal
change in some of the cells: some cells have 2 complete copies of X
chromosomes, some with only 1 and some with 1 complete X and
another altered copy.)

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b. Y chromosome material present in some cells together with the X
chromosome (while some cells have only 1 copy of the X chromosome)
Family history doesn't seem to be a risk factor.

Fig. 185. Turner Syndrome

characteristic
features.

Inborn Errors in Metabolism refers to a group of disorders in which a

single gene defect causes a clinically significant block in a metabolic
pathway resulting either in accumulation of substrate behind the block or
deficiency of the product. These are rare genetic disorders in which the
body cannot properly turn food into energy. The disorders are usually
caused by defects in specific proteins (enzymes) that help break down
(metabolize) parts of food.

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EXAMPLES:
1. Organic acidemias (e.g., methylmalonic or propionic acidemia, multiple
carboxylase deficiency)
2. Fatty acid oxidation defects (e.g., short, medium, and long- chain acyl-
CoA dehydrogenase deficiencies)
3. Primary Lactic Acidoses (e.g., pyruvate dehydrogenase, pyruvate
carboxylase and cytochrome oxidase deficiencies)
4. Aminoacidopathies (e.g., phenylketonuria, hereditary tyrosinemia,
nonketotic hyperglycinemia, maple syrup urine disease [MSUD] and
homocystinuria)
5. Urea cycle defects (e.g., citrullinemia, ornithine transcarbamylase
deficiency, and arginosuccinic aciduria)
6. Disorders of carbohydrate metabolism (e.g., galactosemia, hereditary
fructose intolerance, fructose 1,6-diphosphatase deficiency and the
glycogen storage diseases)
7. Lysosomal storage disorders (e.g., mucopolysaccharidosis, Tay-Sachs,
Niemann-Pick disease, Gaucher’s disease)
8. Peroxisomal disorders (e.g., Zellweger syndrome and neonatal
adrenoleukodystrophy)
9. Disorders of metal metabolism (Menkes syndrome, neonatal
hemochromatosis)

In more details, let us consider some specific IEMs:

1. Phenylketonuria (PKU) is an inherited disorder that increases the levels
of a substance called phenylalanine in the blood. Phenylalanine is a
building block of proteins (an amino acid) that is obtained through the
diet. It is found in all proteins and in some artificial sweeteners.
Classic PKU is the most severe form; it occurs when phenylalanine
hydroxylase activity is severely reduced or absent (fig. 186).
Differences in metabolism between normal and PKU individuals are
shown in fig. 187.

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 Characteristic features include musty
odor in the breath, skin or urine,
caused by too much phenylalanine in
the body, neurological problems that
may include seizures, skin rashes
(eczema), fair skin and blue eyes,
because phenylalanine can't
transform into melanin — the pigment
responsible for hair and skin tone and
abnormally small head
(microcephaly), among others.

Fig. 186. Defective Enzyme system in

individuals with PKU.

Fig. 187. Comparison of

normal individuals
and individuals with
PKU.

PKU is an autosomal recessive disorder. It occurs mainly in people from

Europe and the U.S. The disorder is much less common in Asians and
Latinos. Africa has the lowest rates of phenylketonuria.
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PAH gene:
Cytological location: 12q22-q24.2 [the long (q) arm of chromosome
12 between positions 22 and 24.2]
Molecular location on chromosome (fig. 188): base pairs 102,838,320 to
base pair 102,917,602 on chromosome 12.

Fig. 188. Location of PAH gene on chromosome 12.

2. Maple Syrup Urine Disease

(MSUD) is an autosomal recessive
inherited disorder (fig. 189) in which
the body is unable to process certain
protein building blocks (amino acids)
properly. The condition gets its name
from the distinctive sweet odor of
affected infants' urine and is also
characterized by poor feeding,
vomiting, lack of energy (lethargy),
and developmental delay. If
untreated, MSUD can lead to
seizures, coma, and death. All these
are brought about by build-up of
BCAAs (fig. 190).

Fig. 189. Defective enzyme system of

individuals with MSUD.

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 Fig. 190. Comparison of normal individuals and
individuals with MSUD.

The defective genes are as follows (fig. 191):

a. BCKDHA gene:
Cytogenetic Location: 19q13.1-q13.2
Molecular Location on chromosome 19: base pairs 41,397,788 to
41,425,004
b. BCKDHB gene:
Cytogenetic Location: 6q14.1
Molecular Location on chromosome 6: base pairs 80,106,626 to
80,346,269
c. DBT gene:
Cytogenetic Location: 1p21.2
Molecular Location on chromosome 1: base pairs 100,186,921 to
100,249,862

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 Fig. 191. Chromosomes
where the
genes for
MSUD are
located.
a b c

3. Classic galactosemia (GALT deficiency)(fig. 192-193)

• Galactose-1-phosphate uridyl transferase (GALT) is either missing
or not working properly.
• also known as type I, is the most common and most severe form of
the condition.

Fig. 192. Comparison of normal

individuals and
individuals with
Galactosemia.

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 Fig. 193. Characteristic
health problems
associated with
GALT deficiency.

4. Lactose intolerance is due to lactase deficiency. People sometimes

confuse lactose intolerance with a milk allergy. While lactose
intolerance is a digestive system disorder, a milk allergy is a reaction
by the body’s immune system to one or more milk proteins. An allergic
reaction to milk can be life threatening even if the person eats or drinks
only a small amount of milk or milk product. A milk allergy most
commonly occurs in the first year of life, while lactose intolerance
occurs more often during adolescence or adulthood (fig. 194-195).

Fig. 194. Characteristics

associated with lactose
intolerance.

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 Fig. 195. (A). role of
lactase, (B)
symptoms of
lactose into-
lerance.

Lactose intolerance is
common in adults;
occurs more often in
Native Americans and
people of Asian,
African, and South
American descent than
among people of
European descent.

K. Genetic Engineering and Biotechnology

Genetic Engineering: technology that involves manipulating the DNA of
one organism in order to insert the DNA of another organism, called
exogenous DNA.

Genetically engineered organisms are used to:

• study the expression of a particular gene.
• investigate cellular processes.
• study the development of a certain disease.
• select traits that might be beneficial to humans.
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Biotechnology:
• the use of living cells to make products such as pharmaceuticals,
food and beverages
• the use of organisms such as bacteria to protect the environment
• the use of DNA science for the production of materials for
diagnostics and research

Applications:
• food production (e.g. milk production)
• new horticultural and ornamental plants
• wildlife, aquaculture, natural resources and environmental
management (e.g. bioremediation)
• medicine (e.g. health care, gene therapy, vaccines, medicines)
• forensic science

Organismic biotechnology:
• helps the organism live better or be more productive
• goal: improve organisms and the conditions in which they grow
• study and use natural genetic variations
• example: Cloning How is biotechnology used in everyday life?
Biotechnology helps to meet our basic needs: food, clothing, shelter,
health and safety.
Recombinant DNA (rDNA): the manipulation and combination of DNA
from 2 sources
Examples:
• Bacterial DNA + human gene for insulin
• Plant DNA + bacterial DNA (Agrobacterium tumefaciens)
• Mouse DNA + human DNA = transgenic
The microbe Agrobacterium tumefaciens is harmful to plants and useful to
scientists for the same reason: It transfers DNA into plant genomes. Found
in soil worldwide, A. tumefaciens causes disease in plants by transferring

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its own DNA into plant cells. But in the laboratory, the ability to move all
sorts of genes into plants has made the microbe the standard tool for
investigating plant genetics and modifying crops.

Recombination: Insert a foreign gene into a host

GOAL: to produce many copies (clones) of a particular gene
General procedure in the preparation of recombinant DNA molecule is
shown in fig. 196.
RESTRICTION ENZYME: DNA-
cutting enzymes found in bacteria
(and harvested from them for use).
Because they cut within the
molecule, they are often called
restriction endonucleases.
• Cut gene of interest with
restriction enzyme
• Splice together gene of
interest and vector

To which class of enzymes do the

restriction enzymes belong?

Fig. 196. How to make a

recombinant DNA
molecule.

VECTOR: a DNA molecule used as a vehicle to artificially carry

foreign genetic material into another cell, where it can be
replicated and/or expressed. A vector containing foreign DNA
is termed recombinant DNA.
• Plasmids (fig. 197)
• Viruses
• Particles (DNA coated bullets)
• Exogenous DNA

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 Vectors: Plasmids b

Fig. 197. Plasmid (a) basic parts

(b) bacterial plasmid.

Plasmids can be extracted from the bacterial cell, inserted with certain
genes and re-inserted into the bacterial cell to reproduce and mass
produce the products of the inserted gene.

Vectors: Viruses (fig. 198)

VIRUS: an infective agent that typically consists of a nucleic acid molecule
in a protein coat, is too small to be seen by light microscopy, and
is able to multiply only within the living cells of a host

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Fig. 198. Viruses can carry the gene and insert them into the genome of
the host cell.

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Vectors: Particles (DNA coated bullets) (fig. 199)

Fig. 199. DNA coated bullets introduce

DNA into small host cells.

DNA is introduced into host cells using a particle gun with bullets made of
tungsten or gold and which are coated with DNA. The host cells are raised
in the laboratory. The plant grows and matures with the inserted DNA
already incorporated in its genome.

Vectors: Exogenous DNA (fig. 200)

There is recombination of an exogenous
DNA introduced into eukaryotic cells. The
exogenous DNA comprises a
bacteriophage lambda vector wherein the
vector sequences are important for
specific recombination of the exogenous
DNA into the cellular chromosomal DNA.

Fig. 200. Exogenous DNA introduced to

host cell via a phage.
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bacteriophage: a virus that parasitizes a bacterium by infecting it and
reproducing inside it.

Characteristics of a Vector:
• can replicate independently in the host cell (contains an Ori)
• has restriction sites in the polylinker cloning region [multiple cloning
site (MCS)]
• has a reporter gene that will announce its presence in the host cell
• small in size as compared to the host chromosome for ease of
isolation

APPLICATIONS OF GENETIC ENGINEERING

DNA Recombination can bring a lot of benefits to mankind. Not only are
products directly produced with humans as beneficiaries (e.g. drugs,
hormones), humans also benefit from better crops and cleaner
environment (fig. 201).
Using plasmid as vector, the plasmid that bears the inserted gene is
reinserted into the bacterial cell. The recombinant bacterium reproduces,
reproducing with it the genes of interest. Since bacterial reproduction is
exponential, there is mass production of the gene and from this, specific
proteins are produced in large amounts (fig. 202).

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Fig. 201. Recombinant DNA
technology has
produced many
benefits to humans.

Fig. 202. Here, the product is

insulin which used to
be derived from
pancreas of pigs and
cows.
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Some human genes that have been cloned by recombinant DNA
technology:
• insulin for diabetics
• factor VIII for males suffering from hemophilia A
• factor IX for hemophilia B
• human growth hormone (GH)
• erythropoietin (EPO) for treating anemia
• three types of interferons
• several interleukins
• granulocyte-macrophage colony-stimulating factor (GM-CSF) for
stimulating the bone marrow after a bone marrow transplant
• tissue plasminogen activator (TPA) for dissolving blood clots
• adenosine deaminase (ADA) for treating some forms of severe
combined immunodeficiency (SCID)
• angiostatin and endostatin for trials as anti-cancer drugs
• parathyroid hormone
• leptin
• hepatitis B surface antigen (HBsAg) to vaccinate against the
hepatitis B virus

GENE THERAPY
Some humans are born with defective
genes and if genes that are affected are
essential ones, then abnormalities are
expected. Gene therapy can be done to
correct the specific erroneous gene. Fig.
203 shows two modes of delivery of the
therapeutic gene.

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Fig. 203. Gene therapy.

Animals have been used in various recombination experiments (fig. 204-

205).

What advantages do stem cells have over any other type of cell for
scientific research?

What is Recombinant DNA Technology?

What are the benefits of biotechnology?

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 Fig. 204. Cloning of
sheep.

Fig. 205. Research on Animals: production of transgenic mice.

Transgene: a gene or genetic material that has been transferred
naturally, or by any of a number of genetic engineering
techniques from one organism to another. The introduction
of a transgene has the potential to change the phenotype of
an organism.
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Transgenic mice are popularly being used as models to study obesity,
heart disease, diabetes, arthritis, substance abuse, anxiety, ageing,
Alzheimer's disease, Parkinson's disease as well as different forms of
cancer.
Recombinant genetic technologies are employed to produce organisms
whose genomes have been precisely altered at the molecular level,
usually by the inclusion of genes from unrelated species of organisms that
code for traits that would not be obtained easily through conventional
selective breeding. Example is this frog in fig. 205.

Fig. 205. See-

through frog
(Japan).

To protect plants from pests, genes from a certain strain of bacteria are
inserted into the plant genome, producing a plant that has a built-in pest
resistance. There is no need to use pesticides for these plants (fig. 206).

How is the
understanding of the
processes of
replication,
transcription and
translation an
important
prerequisite to the
understanding of
biotechnology and
genetic engineering?

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Fig. 206. Research on plants: the insertion of gene from Agrobacterium
into a host plant
The popular Bt corn is a product of recombinant DNA technology which
has inserted the gene from Bacillus thuringiensis into the corn genome,
giving the corn resistance or protection from
corn borer (fig. 207).

Fig. 207. Research on Plants. Production of Bt

corn.
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The protein coded by Bt gene turns
toxic in the digestive tract of caterpillars.

Although usually the bacteria are used to generate multiple copies of the
gene that is inserted into its plasmid, bacterial genome can also be
transformed in this manner (fig. 208).

Fig. 208. Research on Microbes.

Bacterial transformation may be referred to as a stable genetic change

brought about by the uptake of naked DNA (DNA without associated cells
or proteins) to increase DNA quantity. Its competence refers to the state
of being able to take up exogenous DNA from the environment.

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Table 11. Genetic Engineering Tools, Processes and Applications.

Genetically engineered organism (GEO) or Genetically modified organism

(GMO): organism whose genome has been engineered in the laboratory in
order to favor the expression of desired physiological traits or the
production of desired biological products

CROPS (fig. 209)

• enhanced taste and quality
• reduced maturation time
• increased nutrients, yields and stress
tolerance
• improved resistance to disease, pests and
herbicides and to drought and heat
• new products and growing techniques
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Fig. 209. Better Crops that result from genetic engineering.

Golden rice (fig. 210) is the result of an effort to develop rice varieties that
produce provitamin-A (beta-carotene) as a means of alleviating vitamin A
(retinol) deficiencies in the diets of poor and disadvantaged people in
developing countries. Because traditional rice varieties do not produce
provitamin-A, transgenic technologies were required.

What kinds of traits have been

engineered into agricultural
crops?

Fig. 210. Golden rice.

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ANIMALS (fig. 211)
• Increased resistance, productivity,
hardiness, and feed efficiency
• Better yields of meat, eggs, and milk
• Improved animal health and
diagnostic methods

Fig. 211. Improved quality of animals and their products.

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ENVIRONMENT (fig. 212)
• "Friendly" bioherbicides and bioinsecticides because plants
produce their own pesticides.
• Conservation of soil, water, and energy
• Bioprocessing for forestry products
• Better natural waste management
• More efficient processing

Fig. 212. Fluorescent zebrafish which were specially bred to help detect
environmental pollutants. By adding a natural fluorescence gene
to the fish, scientists are able to quickly and easily determine
when waterways are contaminated. These fish glow when
certain pollutants are present.
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SOCIETY

1. Increased food security for growing populations (fig. 213)

Fig. 213. More food production to

feed the people.

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2. Production of recombinant pharmaceuticals (fig. 214)

Fig. 214. Recombinant vaccines for prevention of diseases.

POSSIBLE ECOLOGICAL IMPLICATIONS:

• herbicide-resistant superweeds
o If plants have incorporated herbicidal resistance gene, they
themselves could become weeds that cannot be controlled.
• creation of new weeds (herbivore-resistance)
o Since the plants are no longer eaten by pests, they might end
up becoming weeds.
• loss of biodiversity
• reduction of soil quality due to release of toxins from GMO
• harm to beneficial insects
o non-target organisms can become susceptible too.
• creation of new pests
o resistant species might become pests themselves
• sustainable agriculture and organic farming threatened
• crossover of genes to other species (outcrossing)
o the capability of the GMO to escape and potentially introduce
the engineered genes into wild populations
• production of novel allergens and carcinogens

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These concerns have already become a reality in the US, with farmers
complaining that the GM crops that they have raised for some generations
have already lost their built-in resistance to pests and they felt that they
were not able to recover their expenses in the purchase of those GM
strains.
That is why it is important to set into place some regulatory mechanisms
for the use of GMOs.

Regulation in the US
GMOs are regulated pursuant to health, safety, and environmental
legislation governing conventional products.

Assumption: regulation should focus on the nature of the products, rather

than the process in which they were produced.
• US Department of Agriculture regulates transgenic plants.
• Food and Drug Administration regulates the safety of all human and
animal food products in the US (other than meat, poultry, and eggs),
as well as drugs and biological products.
• Environmental Protection Agency regulates pesticidal plants and
genetically-engineered microbial pesticides.
• only require the labels of products which differ from their non-
genetically modified counterparts
Regulation in Canada
Manufacturers and importers who wish to sell or advertise a GM food in
Canada, must submit data to Health Canada for a pre-market safety
assessment, as required under Division 28 of Part B of the Food and Drugs
Regulations (Novel Foods).
- provides assurance that the food is safe when prepared or consumed
according to its intended use.

Regulation in EU
Six Member States currently apply safeguard clauses on GMO
events: Austria, France, Greece, Hungary, Germany and Luxembourg.

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Deliberate Release of Genetically Modified Organisms based on the
precautionary principle.
Applicants who wish to conduct GMO field tests are required the following:
1. application and submission of an environmental risk assessment
2. application to each Member State to market genetically-modified
products
(Each Member State is granted the right to object to such marketing
within their borders.)
The EU and a number of Member States have enacted strict labeling
requirements as well as traceability of GMOs placed on the market.
Labelling will ensure that consumers are given the freedom to choose
whether to consume or not.

Regulation in Australia / New Zealand

• Safety assessment and strict labelling of GM foods.
• All GM food should be assessed regarding safety for human
consumption and approved before sale and use.
• All GM food and ingredients should be labelled where they contain
novel DNA and/or novel protein in the final food, or have altered
characteristics.

Regulation in South Asia

• Regular updating and review of policies governing GMO cultivation
and safety assessment not only to consumers but also to the
environment
• Philippines and Indonesia have approved commercial planting of GM
crops.
CAMBODIA: emphasis on the need to regulate importation of GMO such
as those for contained use, intentional introduction into the environment,
and those for direct use as food or feed or for processing.
• No regulation on pharmaceutical GMOs for humans.

INDONESIA: importation, biosafety and food safety assessment, releasing

varieties (for seeds), and labeling of package products, monitoring for
environment effects
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PHILIPPINES: safe and responsible use of modern biotechnology and its
products as one of the several means to achieve food security, equal
access to health services, a sustainable and safe environment, and
industry development
• no existing law directly addressing biosafety, however, currently in
place are administrative measures (regulations) to address
biosafety-related issues based on existing legislative mandates
(laws)
• no labelling laws
• there are naturally-occurring transgenic crops such as the banana
which has incorporated the genes from the banana streak virus and
the cultivated sweet potato (camote), which contains genes from
the bacterium (Agrobacterium)

Conventional – grown with chemicals and pesticides

Organic – grown naturally – no chemical

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Reasons why LABELLING is NOT implemented:
1. Mandatory labeling would allow consumers to identify and steer clear
of food products that cause them problems.

2. For religious or ethical reasons, many people want to avoid eating

animal products, including animal DNA.

3. Labels on GMO foods would imply a warning about detrimental health

effects, which would stir controversy among millions who strive daily to
maintain or advance their levels of health and wellness through dietary
strategies.

4. Labeling of GMO foods to fulfill the desires of health-conscious

consumers would come at a consequence to all food manufacturers
who use GMO ingredients.

5. Consumers who want to buy non-GMO foods currently have an option

to purchase certified organic foods, which by definition cannot be
produced with GMO ingredients.

6. If GMO foods were segregated from non-GMO foods, the food system
infrastructure (storage, processing, and transportation facilities) would
need to change drastically in a short period of time to accommodate the
need for this change.

7. If anti-GMO activists won the fight to pass GMO labeling legislation, it

would set a precedent to every other highly controversial health topic
in the hands of regulatory agencies.

SUMMARY
First there is DNA, with its two strands which are made up of polymers of
deoxyribonucleotides. It has many conformations depending on the
conditions of the internal enviroment and whether or not the genes are on
or off. Then there is RNA which is made from portions of DNA and is
structurally a lot like DNA except for differences in size, sugar, type of
nucleotides and number of strands. Then we know that proteins are
encoded by the information coming from DNA. There are many genes,
not all of them are expressed at the same time; they are synthesized
depending on the need. All of the processes leading to the synthesis of
DNA, RNA and proteins are catalyzed by various enzymes, some of them
belonging to the same classes.

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Knowing the mechanism of replication can help us fully understand how
repair mechanisms are carried out. Adding to it the understanding of the
mechanisms of transcription and translation which can help us fully
understand how gene expression is regulated. In all of these important
processes, it is amazing how even the non-coding parts of the eukaryotic
genome can be involved in this regulatory process. Also equally amazing
is the fact that there is always a perfect time for everything. Every minute
detail is considered. There is even control at every level, always ensuring
that processes should or should not proceed.
However, not all changes in phenotypes are due to changes in DNA
sequence. There is the complicated concept of epigenetics which can be
traced back to how genes are packaged, whether they can be expressed
or not. If packaging is not done correctly, it can surely create a lot of
problems especially leading to diseases.
However, as part of our evolutionary process, there is mutation that
accounts for a lot of changes in our genome. While the DNA does repairs
there are really those that get away from the repair process. And if the
changes can cause changes in the information passed from DNA, that
could either result to a different product or even cause premature
cessation of transcription and translation. On a bigger scale, mutations
can be as big as a change in the number of chromosome sets, or change
in the number of chromosomes or changes in the segments within the
chromosomes. There are various effects of these kinds of mutations and
they are not necessarily negative. We even enjoy some of the them
especially those that involve food sources. However, we cannot erase the
fact that abnormal chromosome numbers in humans have caused so much
abnormalities and even caused prenatal or early death. Some humans
have inborn errors of metabolism, which are also due to mutations.
The knowledge of the central dogma also helps us in understanding the
principle behind biotechnology and genetic engineering. And all these
have improved our quality of life by providing us with better quality food
products (sourced from both plants and animals) as well as providing us
extra benefits like vitamins, medicines and vaccines which have
prolonged the life span of humans.

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 Reflecting on your Learning (Individual)
After engaging in all learning and assessment activities, reflect on
your learning. Which of the learning outcomes have you achieved?

Fill in the downloadable version of this table. Save as your family name
(section) – ROYL3 and e-mail to your professor. Be sure to follow proper
e-mail etiquette.
What are your key learnings/highlights of
your learning?
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
😊 ~ End of module 3 ~ 😊

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