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[4] ORGANISATION AND CONTROL OF PROKARYOTIC AND EUKARYOTIC GENOME

a) Compare the structure and organisation of prokaryotic and eukaryotic genome

Features Prokaryotic genome Eukaryotic Genome


1) Genome size 0.6Mb to 10Mb Can range from less than 10Mb to 100
000Mb
2) Gene Length Shorter than that of Eukaryotes. Even Longer
with the introns removed from the latter,
the prokaryotic gene is only 2/3 the
length of the eukaryotic gene
3) Coding region Continuous from beginning to end with Discontinuous, interrupted by introns
no interruptions by non-coding
sequences
4) Cluster By operons Operons absent.
organization of Structural genes involved in the same Exist in multi-gene families with each
genes pathway are controlled by a single gene controlled by its own promoter and
promoter and operator. operator.
May occur at same chromosomal locus
or at many loci.
May not be regulated together
5) Form Single, Circular, double stranded Two or more linear, double stranded
Chromosome (‘misnomer’ as it is not chromosomes.
associated with histones) Possess smaller mitochondrial genomes.
Additional smaller circular chromosome, In plants, third genome is found in
plasmid-carry genes not essential for chloroplasts
survival E.g antibiotic genes
6) Location Found in the nucleoid, not membrane- Found in the nucleus (mainly), bounded
bound by nuclear membrane
7) Amount of non- Little, limited to promoter and operator A lot, more than 90% of genome is made
coding DNA (regulatory sequences) up of non-coding DNA such as satellite
DNA, introns etc
8) Repetitive Infrequent repetitive sequences but >58% of genome is made up of repetitive
sequences nothing close to the genome wide sequences which include telomeres,
repetitive sequences found in the transposable elements
Eukaryotic genome
9) DNA Genome is longer than the prokaryote. Double-stranded DNA is coiled slightly
Organisation Thus genome has to be folded to fit into less than 2 turns (146 bases) about
the prokaryote. This is done by histones to form nucleosomes with
supercoiling DNA and complexing it with remaining DNA forming linker DNA.
DNA binding proteins. These nucleosomes will then be coiled to
form a 30 nm solenoid fiber structure. It
is stabilized by H1 histones.
This 30 nm structure will then be
attached to a central protein scaffold
where it will loop out from and fold
further to form the 700 nm Metaphase
chromosome.
10) Supportive Held together by DNA binding Proteins Held together by H1 histones and ionic
structure bonds
b) Describe the structure and function of the portions of eukaryotic DNA that do not encode for protein or
RNA
c) Describe the role of telomeres and centromeres
Structure Function
1) Promoters Required for identification of correct DNA template strand for transcription. (TATA
box)
Site where RNA polymerase attaches
In Eukaryotes, general transcription factors will assemble at the promoter to form
transcription-initiation complex so that RNA polymerase can bind.
2) Introns Non-coding DNA found in between exons. Enables a single gene to give different
polypeptides. Important in Alternative Splicing.
3) Repeating Microsatellites: short repeating sequences that can be use in DNA fingerprinting
Sequences Transposable elements: Alu Element that is associated with certain diseases and can
track evolution.
4) Control elements Upstream Regulatory elements (UREs), Enhancers, silencers
Short non-coding DNA sequences that interact with activator proteins or repressor
molecules to either increase or decrease the rate of transcription.

5) Stop codons: Signals to ribosome to stop translation


UAG, UGA, UAA
6) Centromeres  Sister chromatid adhesion
 Kinetochore formation
 Control of gene expression
 Pairing of homologous chromosomes
7) Telomeres  Postpone the end-replication problem by acting as a buffer so that
important genes are not lost
 Maintain integrity of chromosomes by preventing DNA-repair machinery
from annealing together the ends of broken chromosomes
 Determines cell lifespan as after a few rounds of DNA replication and a
critical length of telomeres is reached, the cell will then undergo apoptosis.
Also serves to prevent accumulation of DNA damage.

d) Describe the process and significance of gene amplification

Definition: Gene amplification is the result of repeated replication of DNA in a limited portion of the genome, in
the absence of or to a much greater extent than replication of DNA composing remainder of genome.

Process:

1) Slippage of DNA template strand relative to new strand being synthesized thus resulting in a particular
region being transcripted twice.

2) Unequal crossing over during Prophase 1. This leads to further pairing problems in subsequent divisions
and eventually results in a string of the same gene in a row

3) Naturally via excision of a repeating unit and its extra chromosomal replication in a plasmid (E.g. Yeast)
4) Via the transcription of a gene to get the mRNA and then reverse transcription of this mRNA to obtain an
extra DNA copy of the gene.

5) Breakage and rejoining

Significance:

-Development (amphibians)

 Oocytes (eggs) are large, about a million times larger than a normal somatic cell. Has to support a large
amount of protein synthesis. Hence, requires a large amount of ribosome and rRNA. This need is thus met
by amplifying the genes that code for rRNA. The oocyte contains about 200 such genes and these are then
amplified 2000-fold to obtain about 1 million copies of the gene.

-Cancer

 Amplification of proto-oncogene may result in it becoming an oncogene which will then continuously
code for products that stimulate cell to divide
 Amplification of Multi-Drug Resistance (MDR) gene. Important in the formation of drug-resistant tumours

-Evolution

e) Describe the Eukaryotic processing of pre-mRNA

1) 5’ capping
-Removal of the 5’ phosphate group and replacing it with a Guanine residue that is later methylated using
methyl transferase to form 7-methylguanosine cap
-Adjacent nucleotides are also methylated
-Done to protect the 5’ mRNA end from exonuclease degradation and the 5’ cap also helps in initiation of
protein synthesis
2) Cleavage and Polyadenylation
-GU-rich sequence and polyadenylation signal sequence.
- Cleavage factors recognize these sequences and will cleave in the middle of
the two sequences
-Poly(A) polymerase will then add 250 A residues to the new 3’ end and then
stabilize it with proteins to form a poly (A) tail
-This protect the 3’ end of the mRNA from exonuclease degradation
3) RNA splicing
-A complex of small nuclear RNAs associated with proteins will form a small
ribonucleoproteins (SNURPS) that when associated together will form a
splicesosome.
- The splicesosome will then loop out introns and remove them
- It will then ligate together upstream and downstream exons.
4) Alternative Splicing
-This is whereby exons are cut and rejoined together in different ways so as to give
rise to different polypeptides.

f) Define control elements and explain how they influence transcription.


Definition: Control elements are non-coding DNA sequences that interact with specific transcription factors (E.g.
activators and repressors) so as to determine the beginning and end of a transcript as well as the rate of
transcription.
For enhancers (dixtal control elements):
 They interact with activator proteins
 DNA bending protein will then result in the protein looping out so that activators bound to enhancer can
interact with transcription-initiation complex on promoter to speed up its assembly.
For silencers:
 Repressors will bind to it thus decreasing rate of transcription
For UREs (proximal control elements):
 Interact with activators to increase the rate of transcription

g) State the various ways in which gene expression may be controlled at translational and post-translational
level
Eukaryotes-translational control
 By natural mRNA-degradation machinery
-Removal of 5’ cap
-Exonuclease will start nibbling at the mRNA in the 5’ to 3’ direction
 By lifespan of mRNA
-The longer the lifespan of the mRNA, the more times it can be translated and vice versa
 By microRNAs
-RNA molecules can bind to 3’-UTR and thus either prevent translation or trigger destruction of the mRNA
 By blocking initiation of translation
-prevent binding of RNA polymerase
Post-translational control
 Chemical Modifications: Hydroxylation (Hydroxylation of proline to form hydroxyproline), Acetylation
(histone acetylases), Glycosylation, Phosphorylation (phosphorylation cascade), Methylation
 Cleavage to yield functional proteins from polyprotein (E.g. ubiquitin)
 Protein Degradation-Destruction of polypeptides that are damaged or have an inherent unstable C-
terminal by binding it to ubiguitin which will then break it down before being released to be reused

Prokaryotes-translational
 Block the promoter sites for some cistrons to prevent RNA polymerase from binding so as to prevent
translation of some cistrons relative to others
 Protect mRNA from exonuclease degradation so as to allow the mRNA to be translated more times
 For some prokaryotes, the tertiary structure of the mRNA resembles the shape of the product coded for
by the mRNA. Thus, when there is too much of the product, the product just binds to its own mRNA thus
stopping its translation
 Formation of mRNA duplex near the promoter site to prevent RNA polymerase from binding
Post-translational
 Methylation of native DNA allows bacteria to protect themselves from foreign DNA attack
 Phosphorylation of glycerol kinase in Streptoccocus Faecalis serves to deregulate glycerol degradation

h) Outline the differences between prokaryotic control of gene expression with the Eukaryotic model
Features Prokaryotes Eukaryotes

1) Chromosomal control Absent as chromosome is not DNA is unwound using histone


associated with histones acetylases while chromatin
remodeling complexes displace
nucleosomes

2) Transcriptional control By operons By control elements such as


Structural genes involved in the promoter, enhancers, UREs and
same pathway are controlled by a silencers.
single promoter and operator Enhancers, UREs and silencers
interact with activators and
repressors to influence the rate of
transcription.
There is also combinatorial control
whereby varied sequence of control
elements for each gene is more
important than a unique control
element and coordinate control

3) Post-transcriptional Absent as both transcription and Present, includes 5’ capping, cleavage


control translation take place and polyadenylation, RNA splicing and
simultaneously due to absence of alternative splicing
nuclear membrane
4) Translational control  Block the promoter sites  By natural mRNA-
for some cistrons to degradation machinery
prevent RNA polymerase -Removal of 5’ cap
from binding so as to -Removal of poly(A) tail
prevent translation of -Exonuclease will start
some cistrons relative to nibbling at the mRNA in the
others 5’ to 3’ direction
 Protect mRNA from  By lifespan of mRNA
exonuclease degradation -The longer the lifespan of
so as to allow the mRNA the mRNA, the more times it
to be translated more can be translated and vice
times versa
 For some prokaryotes,  By microRNAs
the tertiary structure of -RNA molecules can bind to
the mRNA resembles the 3’ UTR and thus either
shape of the product prevent translation or trigger
coded for by the mRNA. destruction of the mRNA
Thus, when there is too  By blocking initiation of
much of the product, the translation
product just binds to its -prevent binding of RNA
own mRNA thus stopping polymerase
its translation
 Formation of short
antisense RNA molecules
that form duplexes near
the binding site of
ribosome thus inhibiting
translation

5) Post-translational  Methylation of native  Chemical Modifications:


control DNA allows bacteria to Hydroxylation (Hydroxylation
protect themselves from of proline to form
foreign DNA attack hydroxyproline), Acetylation
 Phosphorylation of (histone acetylases),
glycerol kinase in Glycosylation,
Streptoccocus Faecalis Phosphorylation
serves to deregulate (phosphorylation cascade),
glycerol degradation Methylation
 Cleavage to yield functional
proteins from polyprotein
(E.g. ubiquitin)
 Destruction of polypeptides
that are damaged or have an
inherent unstable C-terminal
by binding it to ubiguitin
which will then break it
down before being released
to be reused

(i) Describe how proto-oncogenes are converted into oncogenes including gain-of-function mutations in
proto-oncogenes and loss-of-function mutations in tumour suppressor genes
Definitions:
Proto-oncogenes are genes that code for products that stimulate normal cell division.
Oncogenes are mutated versions of proto-oncogenes that bring about uncontrolled cellular division.
Tumour suppressor genes code for proteins that suppresses cellular division.

Proto-oncogenes are converted to oncogenes through a gain-of-function mutation whereby only one
allele needs to be mutated for it to become an oncogene. It is a gain-of-function mutation as the gene has
acquired an additional ability which is to continuously transcribe proteins.

This is done via:


a. Translocation, whereby DNA breaks and then rejoins at another position. If this occurs in the
region controlling the production of the protein, there will be overproduction of the protein. If
this mutation occurs in the protein-coding region however, an overactive protein may be
produced.
E.g Chronic Myelogeous Leukemia (CML)- caused by the breakage of chromosome 7 and
translocation of the broken part to chromosome 22. This results in an abnormal Bcr-Acl gene that
codes for tyrosine kinase receptor that is always activated regardless of the presence of growth
factor. As a result there is excess production of myelocytes, a type of white blood cell resulting in
CML

b. Point mutation-chemical change in the bases. Can occur in either the protein- coding region or
the region that controls transcription and translation of the protein
E.g. Ras oncogene- Bladder cancer has been linked to the ras oncogene. A point mutation in the
gene on chromosome 9 that resulted in the substitution of a Thymine for a guanine thus
resulting in a change of amino acid to Valine resulted in the oncogene. This results in the G-
protein coupled receptor that is coded for by this gene to be unable to release GTP and cycle to
the inactive form. Thus, it will continuously send signals thereby resulting in overproduction of
the protein and uncontrolled cell division

c. Gene amplification of proto-oncogenes


d. Insertional Mutagenesis
-E.g Human papillomavirus
-the virus can insert its genome into the chromosome of the host cell as a site near a proto-
oncogene. As such, the proto-oncogene comes under control of the viral promoter and
enhancers. Thus, since viral promoter and enhancers are not controlled by the same
environmental factors, this may result in the proto-oncogene being translated at a higher rate
than normal.

Tumour suppressor genes will undergo a loss-of-function mutation when both alleles are mutated. This is
because when the gene is mutated, it loses its ability to code for a protein that can suppress cell division.

For some individuals, they may already have a copy of the mutated allele as they inherit it from their
parents. However, they will remain phenotypically normal since they still have one copy of the normal
allele. But, later on in life, the normal allele may be lost and the phenotype of the mutated allele will
surface. This occurs via Loss of Heterozygosity.

Loss of Heterozygosity can occur via:


a. Deletion of normal allele
b. Deletion of chromosomal arm containing normal allele
c. Deletion of chromosome containing normal allele
d. Non-disjunction during mitosis
e. Deletion of normal allele followed by duplication of mutated allele.
f. Genetic recombination

(j) Explain how mutations in tumour suppressor genes can contribute to cancer and describe the development of
cancer as a multi-step process.

Using p53 gene to explain:


Tumour suppressor genes such as the p53 gene code for transcription factors. Thus under normal conditions, the
p53 protein will bind to the mdm complex which inactivates it. However, in times of stress, a series of reactions
will be kicked off and the p53 protein will dissociate from the mdm. Thereafter, this protein can then bind to
several genes to stall the cell cycle so as to allow time for DNA repair proteins to repair damaged DNA. Essentially,
these proteins are able to:
1) Hold the cell cycle to allow time for DNA repair proteins to repair DNA damage
2) Recruit DNA repair proteins
3) Trigger cell apoptosis if DNA damage is irreparable
Thus, through these, the protein is able to prevent cells with DNA damage from proliferating. However, if they are
mutated such that they lose their function, cells with DNA damage can then proliferate and since their DNA is
damaged and inherently unstable, it will undergo more mutations as it divides until eventually it is completely
deregulated and is dividing uncontrollably resulting in cancer.

The development of cancer is a multi-step process as it involves the sequential acquisition of gain-of-function
mutations of proto-oncogenes to form active oncogenes and loss-of-function mutations in tumour suppressor
genes.

About 6 mutations must occur at DNA level for a cell to become fully cancerous.

The development of cancer results from a single cell which has accumulated mutations over a period of time.