These are comprehensive lecture notes for the BIOC0005 Week 7-10 lectures. BIOC0005 is taken by 2nd year BSc students studying Molecular Biology, Biochemistry and other life sciences degrees. Important knowledge/exam points are highlighted or typed in red. I received a first honours for this course...
One genome = 23 chromosomes (22 autosomes, 1 sex chromosome)
• Each chromosome has 50-250Mb of DNA
• 3*10^9 bp per genome.
• Somatic cell (2 genomes = 6*10^9 bp)
• Very long and tightly packed.
There is also another genome - mitochondrial genome
1.5% of genome are exons. 25% are introns. 43% are repetitive DNA considered non functional.
Other DNA: regulatory regions of genes (ie. promoter), pseudogenes, fragments of genes.
Protein coding genes are split into introns and exons.
• Introns are spliced out after mRNA is made to make the processed mRNA.
• In the DNA, protein coding genes have control elements at the 5' and 3'
◦ 5' flanking sequence: promoters, enhancers
◦ 3' flanking sequence: enhancers.
In eukaryotes, RNA polymerase requires help from enhancers and other proteins to bind to the promoter.
Processed mRNA
= have polyA tail and introns removed.
• This leaves the nucleus and goes to the cytoplasm.
Protein coding genes are different in size.
• Some are larger some are smaller.
• Largest gene is Dystrophin.
• in different genes, they have different % of exons. (Ie. tRNA gene is 100% exon, dystrophin is 0.6% exon)
• Smaller genes tend to have a larger percentage of exons.
Gene families
• Sets of genes where base sequence is similar.
• Thought to arise by gene duplication, and genes acquire some base changes. (They code for things, so it is not
'repetitive dna'
• Encode proteins with different properties.
• Example: Globing gene family code protein chains for haemoglobin.
• Cluster means a number of genes that are related in sequence.
Alpha globin gene cluster on chromosome 16 ( a family)
Beta globin gene cluster on chromosome 11 ( a family)
• The genes code for similar sequences but occur at different stages of development.
• Ordered in the order of when they are needed.
Histone Gene family
• H1, H2A, H2B, H3, H4 are histones needed for nucleosome for DNA packaging.
• They make an octamer of 1 H1, and 2 of the rest.
• 140bp of DNA wraps twice around the histone octamer.
• Histone genes have no introns and polyA because need to be made quickly.
• 11 histone gene clusters and 60 histone genes distributed over 7 human chromosomes. Although duplicated,
each member of a particular histone gene family encodes the identical protein (same one even though there
are some changes!)
Gene duplication outcomes
• 3 outcomes
• Selective pressure on both genes: genes stay similar and functional ie. in globin gene family.
• Selective pressure on one
◦ Pseduogene = one copy become non functional
◦ One copy acquires a new function.
,Part 2
Pseudogenes
• Resemble a functional gene but do not encode functional RNA or protein.
• Different types of pseudogenes from different processes
◦ Type 1 pseudogene - Gene duplication and lots of mutations makes it non functional/inactive.
◦ Unitary psedogene - there was only one of this gene, it becomes mutated and useless. It is not part of a
gene family. Mutation is tolerated (not lethal).
◦ Processed pseudogene - these come from mRNA. mRNA reverse transcribed to cDNA by viral reverse
transcriptase and integrated into chromosome (since mRNA was processed, it has no introns and 5' and 3'
elements)
‣ Features: long string of A (polyA tail).
‣ No promoter and no introns.
Other genes - fragments and truncations
• Functional gene can be truncated at either 5' or 3' end.
• Gene fragments are short isolated regions f genes.
◦
◦
Part 3 repetitive DNA
• 43% of genome.
• 4 classes: LINES, SINES, LTR elements, DNA transposons.
Nonretroviral retrotransposons
LINES (L1) - long interspersed nuclear elements
• There are different classes of LINES, one is L1
• L1 elements are the only autonomously active family in humans
◦ Can copy themselves and reinsert a copy at a different site in human chromosome.
• Individuals vary in L1 insertions because L1 randomly inserts themselves.
• L1 is dynamic and gives plasticity to the genome.
• Full length L1 element is ~6000bp (long).
L1 element has
• Promoter
• ORF1: binds both RNA and DNA and acts as a chaperone
• ORF2: encodes a poly protein with reverse transcriptase and endonuclease activity. (This helps LINE have
autonomous activity)
• polyA stretch.
LINES promote their own transposition.
• Promoter -> transcription
• Reverse transcriptase -> turns LINE RNA
back to DNA.
• Endonuclease - cleaves target
chromosomal DNA.
How does LINE copy itself and reinsert into
the genome?
1. LINE is transcribed.
2. Transcribed mRNA goes too cytoplasm
to be translated. Makes the proteins of
ORF1 and ORF2.
3. ORF1 is a chaperone and binds ORF2
(reverse transcriptase) and LINE
mRNA and brings it back to the nucleus.
, 4. The target site for attack on the chromosome is a AT rich region.
5. LINE mRNA anneals to the target T rich region by its polyA tail.
6. Endonuclease (encoded by ORF2) cleaves the target site. 3' OH group on cleaved chromosome is primer end
for synthesis of cDNA molecule using LINE mRNA as template.
7. Target site primed reverse transcription: Reverse transcriptase (encoded by ORF2) makes CDNA using mRNA
template.
8. Human RNase H is an enzyme that cleaves phosphodiester bonds in an RNA that is hydrogen bonded to DNA
A. In this case, mRNA is h-bonded to cDNA.
B. It will cleave the mRNA bonds. mRNA is degraded. Now there is a single cDNA strand.
9. DNA polymerase can replace the RNA with DNA using exonuclease and synthesis activity. Now a double
stranded DNA is made.
10. LINE DNA is inserted in human genome.
Consequences of LINE insertion into genome
• Can disrupt genes by inserting in exons.
• Insertion into promoter region can silence transcription of a gene.
• Can insert into introns and slow down transcription
• Can cause diseases.
• LINE insertion can cause differences between species (ie. human line insertion into FMO1 gene cause it to be
inactive, while it is expressed in all mammals)
Most LINES are truncated at 5' ends, suggest reverse transcriptase did not fully transcribe the mRNA into cDNA.
(Synthesis in 5' to 3' direction, so 5' end of RNA is not transcribed)
• Promoter is at 5' end of mRNA, so truncated lines are not transcribed (no promoter).
Around 100 copies of LINES are full length (these can be transcribed)
• However, tend to be heavily methylated (silenced).
SINES (Short interspered nuclear elements)
• Also transposes to insert into genome but does not code for reverse transcriptase itself.
• Most abundant SINE is the Alu repeat family.
◦ Short - 280 - 300 bp in length.
‣ Primate specific
• Features of Alu repeat
◦ Have internal promoters.
◦ But there are A and B binding sites for RNA polymerase 3.
• SINE DNA transcribed to mRNA by human RNA polymerase 3.
• mRNA is reverse transcribed to cDNA by reverse transcriptase from L1 element.
• Integration into chromosome like LINE.
SINEs, like tRNAs and many small-nuclear RNAs possess an internal promoter and
thus are transcribed differently than most protein-coding genes.
• RNA polymerase 3 is a enzyme that transcribed genes with internal promoters.
Part 4 improving healthcare
• First human genome sequence provides reference template.
• Have a template to put genome back together is easier.
• Identifiy variations such as Snps, insertions, deletions.
• Pharmacogenetics or pharmacogenomics - tailor drug treatment to individuals according to genetic makeup.
◦ Aim: improve efficacy and avoid adverse drug reactions.
◦ Example: drug metabolizing enzymes such as Cytochrome P450 (CYP family) detoxify drugs and aid
excretion. Can also activate some drugs into the active form.
‣ Lots of mutations in the CYP2D6 gene cluster than can cause no enzyme to be made (PSEUDOGENE).
These mutations affect drug clearance.
‣ Some mutations lead to not being able to clear the drug -> can be lethal.
‣ Poor metaboliser: Others cannot convert the inactive molecule to active molecule (cannot convert
codeine to morphine and therefore has no effect).
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