Sequence Organisation Flashcards
What percentage of the genome is coding?
20’000 coding genes but that only represents 1.1% of the genome. There are also 12’000 pseudogenes. Genes are not evenly distributed within chromosomes. GC content is much higher within gene rich areas and CpG islands usually found just before or in promoter regions.
What is G banding?
Treat with trypsin then stain with giema. G bands represent dense low transcription heterochromatin with low GC and CpG islands and high L1 elements. R bands represent high transcription, high GC and CpG content with increase Alu elements. C bands are highly heterochromatic containing long satellite arrays.
L1 elements are AT rich whilst Ale elements are GC rich.
What are mobile elements?
These are stretches of DNA that can or used to be able to move around and copy themselves. They make up 45% of the genome. 41% are retrotransposons
What are DNA transposons?
These make up 3% of the genome and encode a transposase flanked by inverted repeat sequences. However the transposase lost its ability to move about 37mya. Most common is MER making up 2.4% of the genome.
What are LTR retrotransposons
These are autonomous retrotransposons with long terminal repeats and make up 8% of the genome. These are endogenous retroviruses the best example being HERV. HERV has a speciifc structure: LTR-Gag-Pol-Env-LTR. In humans the env is inactive so they are no longer able to move.
Throughout the genome there are many solo LTRs which can act as promoters to genes.
What are LINE retrotransposons
These are also autonomous but are termed long interspersed nuclear elements making up 20% of the genome. L1 is the most common type making up 17% of the genome. L1s are AT rich and arose 100mya. It is flanked by short direct repeats and not LTRs. The 5’ UTR contains a bi directional promoter meaning it can effect nearby genes. Within the UTRs it has two ORF.
There are 0.5m copies in the genome but 95% are truncated at the 5’ end and so average only 900bp. Most full length have mutations and so aren’t active. Some L1s still active and are termed hot - Ta1 family.
How do we test the activity of different L1 families?
Insert a reversed and so inactive antibiotic resistance gene containing an intro into the construct and into a plasmid. If it is transposed the intron will be removed and the gene reverse transcribed into a functional antibiotic resistance gene.
How did we characterise the process involved in L1s reverse transcription?
Using the same construct used to test which families were active allowed characterisation of what the different ORF genes did.
ORF1 forms trimer that binds to L1RNA and ORF2 protein. ORF2 has both nuclease and reverse transcription function. This creates a nick and the 3’ end of genomic DNA binds to poly A tail of L1. RT occurs then. This is termed target primed reverse transcription - TPRT becasue the endonuclease target site results in a short repeat at either end of the new L1.
This process is easily interrupted hence why it is often truncated.
What are non autonomous retrotransposons
SINE’s - small interrupted nuclear elements making up 13% of the genome. Main example is Alu which accounts for 11% of the genome. There are 1m copies and evolved from 7SL RNA gene 80mya which is the RNA component of the signal recognition particle (SRP) which recognises newly synthesised proteins.
Alu elements evolved through 2 steps - deletion of the s domain then amplification and dimerisation.
Structure = left and right monomer seperated by a PolyA and flanked by 2-18bp direct AT rich repeats. Left monomer contains important A and B motifs.
How do Alu elements retrotranspose?
Why is Alu found in GC rich regions but L1 in AT rich?
How successful is Alu
Alu RNA 3D structure binds to signal recognition particales SRP9 and 14 and becomes associated with the ribosome. If an L1 element is being translated then the ORF2 associated with the Alu RNA sequence.
Insertion is actually random but there has been selection for loss from certain regions over time.
Alu much more successful than L1 and there was a large expansion about 40-65mya with 1 expansion per primate birth at that time.
What is an SVA elemetn?
SINE + VNTR + Alu. VNTR = variable number of tandem repeats. There are 3000 copies in the genome. Hominid specific version arose 25mya and is non autonomous relying on L1.
How is L1 mediated retrotransposons controlled?
L1 retrotransposition occurs in all cell types. Al1 1 in 20 births, L1 1 in 100 and SVA 1 in 900. This is regulated with epigenetic modification of CpG islands, RNA and DNA binding proteins and premature Poly A of L1 mRNA.
What consequences can retrotransposons have?
Deleterious mutations can occur but if in somatic - no problem. Some examples where this has occured in the germline are: DMD and alpha thal. In somatic retrotransposons could cause cancer by activation onco gene or disrupting a tumour supressor gene.
Retrotransposons is a very minor contributor to mutations in our population.
L1 in introns can change splicing and also splicing proportions e.g. major vs minor product. ORF2 particularly involved in this.
How can retrotransposons effect gene expression?
Promoter activity can be modulated by HERV LTRs
Premature adenylation can prevent gene expression
Exonisation by moving an Alu element into a gene
Spread of heterochromatin due to decrease in GC rich and CpG via L1.
How can retrotransposons result in gene duplication and evolution?
Non homologous crossing over events via retrotransposons on different loci can lead to duplications/deletions of genes. IN the event of a duplication this new gene may acquire mutations and evolve anew function.
This process can also lead to large genomic disruption.
