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