Lecture 20/21 Flashcards
transposable elements are _______ in large genomes
transposable elements are ABUNDANT in large genomes
are genomes large because they have TE or do genomes have TE because they are large?
how do they know?
genomes are large because they have TE
compare closely related plants –> ones with larger genome has more TE
Where are transposable elements found? what does this indicate about how they affect genes?
in INTRONS/intergenic regions, therefore not disrupting genes very much
why are transposable elements found mainly in introns?
would be lethal if they are in exon –> the TE are not targeting themselves to introns, just being negatively selected when inserted into exons
when can introns insert into genes? consequence?
1/600 spontaneous mutations causing human disease are due to transposition of TE in gene –> not persistent over time, just appear in an individual
3 ways that TE affect genome
- insertion in gene induces LOF
- inserion disrupts splice potential
- TE mobility destabilizes genome to cause cancer
why are TE helpful?
allow for phenotypic novelty and adaptive change
describe the dysgenic cross of drosophila
female lab strain X male wild-caught strain
F1 are normal but germline cells don’t develop so no F2 can develop
why do the germline cells not develop in dysgenic cross?
mobile P element in male wild-caught strain causes genome instability leading to mutation, abnormal chromosomes, and non-disjunction
describe the reciprocal cross of drosophila
male lab strain X female wild-caught
F1 normal, germline cells normal, F2 normal
what strain carries the P element?
wild-caught
what can we conclude from the fact that the germ cells are normal despite the wild-caught strain carrying the P element?
there must be a mechanism in FEMALE wild-caught that blocks P element mobility and preserves genome integrity
what is the result of male wild-caught X female wild-caught? why?
everything normal –> bc female wild-caught is present
describe the TE studied in C. elegans
Tc1 is class 2 TE that autonomously encodes transposases
why do we want to use Tc1 to study TE mobility?
Tc1 elements only transpose in somatic cells, not germline cells –> therefore must be ‘silencing machinery’ that represses Tc1 mobility
why is it important that the silencing machinery is encoded by genes?
we can KO genes and perform a genetic screen
describe the genetic screen performed in C. elegans
randomly KO genes
- if the gene silences mobility: the KO will make Tc1 jump out of Unc22 and worm will be smooth-gliding
- if the gene is not involved in silencing: Tc1 will stay in Unc22 and worm will be twitching/uncoordinated
what allows us to see effects of Tc1 in progeny?
Tc1 mobility is affected in germline cells, so the mutations are affecting germline cells and we can see effects in progeny
is Tc1 silenced in the smooth-gliding worm?
no, Tc1 can jump out and give proper Unc22 function
what pathway were many genes that block Tc1 mobility involved in?
genes involved in RNAi silencing pathway
RNAi silencing pathway (5)
- dsRNA precursors processed into short ssRNA
- processed by dicer
- bind to RISC
- ssRNA-RISC complex recognize mRNA targets via complementarity
- promotes mRNA degradation or represses mRNA translation (i.e. silencing)
2 types of short ssRNA that bind RISC
- miRNA
- siRNA
miRNA vs siRNA
miRNA is endogenous gene
siRNA is foreign gene (transgene, virus, TE)
describe Tc1 with the RNAi silencing pathway
- Tc1 inserts right next to gene
- both get transcribed, including Terminal Inverted Repeats flanking the transposase
- forms complex with RISC that can recognize Tc1 anywhere in the genome and silence it
Tc1-RISC complex is an example of:
Tc1-RISC complex is an example of genome surveillance
what is genome surveillance?
cellular strategy to detect and prevent transposable element mobility
describe the genome surveillance pathway in drosophila
TEs in “pi cluster” form piwi-argonaute complex that surveys the genome
describe the piRNA silencing pathway in drosophila if TEs are in the pi-cluster
- TE processed into small piRNA
- piRNA joins argonaute and surveys
3 possibilities of piRNA silencing pathway if TEs are not in the pi-cluster?
- TE is inactive and not transcribed
- TE is older so it is also found in pi-cluster, so can be silenced
- TE is newer so it has not jumped into pi-cluster yet, so there is no programmed PIWI and transposase can be made (not silenced)
how are piRNAs similar to siRNAs?
piRNAs are short ssRNA that interact with a protein complex to destroy complementary target mRNAs
how are piRNAs different from siRNAs?
piRNAs don’t start out as dsRNA but as long mRNAs from pi cluster
now that we know about the piRNA silencing pathway, how can we explain the dysgenic cross?
P-element in male wild-caught strain can mobilize and cause genome instability –> no germline cells
now that we know about the piRNA silencing pathway, how can we explain the reciprocal cross?
P-element in female wild-caught strain is in pi-cluster so PIWI can block mobility and germ cells are normal
what is p53?
tumour suppressor
4 functions of tumour suppressors when they are WT
- cell cycle regulator
- checkpoint control
- apoptosis
- DNA repair proteins
what happens when p53 is mutated?
tumour!
what is a hypothesis for how p53 mutation causes tumour?
p53 is involved in silencing TE mobility, so mutant p53 can affect genome stability
describe TE expression in Wilms tumour with mutant p53
increased LINE ORF1p expression
what is ORF1p?
marker for mobility of LINE1
what is p53 required for?
transposon mobility in Drosophila
what happens if there is loss of p53 aka p53 mutation?
loss of p53 = deregulation of retrotransposon activity = mobility not silenced –> tumour
in p53-/- mice, what happens if you give drosophila transgenic WT p53?
mobility is silenced
in p53-/- mice, what happens if you give human transgenic WT p53?
mobility is silenced
in p53-/- mice, what happens if you give human transgenic mutant p53?
mutant p53 can no longer block TE mobility so mobility NOT silenced
what is an issue with the p53 study?
these assays were done in isolation from other components of the tumour
