Download doc Genetics 03-22

GENETICS – 03-22-2010
Slide: Hybrid dysgenesis is the result of DNA Transposons (P elements)



P elements of Drosophila
Are also nonautonomous versions with internal deletions.
There are defective p elements that can transpose on their own but can with a
complete P element.
 P repressor and transposase are a result from alternative splicing of the same
gene- splicing of P element is tissue specific –
 Spliced different in somatic cells and germ line cells – Entron 3 is spliced out and
a longer proteine of 87KD is made  Both the repressor and the transposase are the products of the same gene.
 P element is one of these class 2 type DNA transposon – incredibly valuable for
molecular bio – allowed developmental genetics for fruit flies.
 It can be used as a tool for mutagenesis – because they produce a tag of a known
DNA sequence and therefore easy to identity the gene that’s causing mutation
because this known DNA sequence is sitting in the middle of it.
 Approach called Transposon tagging.
Harnessing of Eukaryotic Transposable elements
 P elements can also be used for introducing Genes back into Drosophila – used
carrier to insert your gene of interest into the germ line of dysgenic flies –
progeny will express that gene.
 Originally you needed to inject two plasmids into the germ cells of the dysgenic
flies.
 Progeny will reflect the expression of that Gene.
 Tricks now to use different genetic vectors to express the gene in any kind of
cell/tissue that you’d like.
Transposable elements & host genomes:
 Why do organisms have these?
 Maybe they are simply selfish DNA capable of self-replication and that’s a
necessary and sufficient explanation.
 But the transposable elements largely explain a long-standing paradox called the
C-value paradox – the Genome size doesn’t really correlate with the complexity
of the organism or with the number of genes. – Table of various organisms and
genome size (protein-coding genes) and the number of predicted genes.
 Number of genes and size hard to argue it correlates well with complexity of
organism
 What does correlate is that the larger genomes full of transposable elements.
Transposons & the human genome:
 So for us – roughly 45 percent of genome composed by transposable elements –
20-40 thousand of LINe elements
 SINEs are very short but 20-30 thousand in our genome.
 Roughly 300 0000 DNA transposons –
How can so many Transposons be maintained?
 What do all these things do and how do they avoid replicating totally out of
control/killing their host – but if the host dies then the transposable elements die
too.
 Most transposons are dead – dead because either they have a mutation in their
transposase genes and they also have mutations in their flanking repeats – they
can’t hop anymore –
 A lot of transposons are inactive –capable of mobility but kept in one place by
repressors. Those transposons can be activated under certain conditions – could be
advantageous for the organism because it could induce rapid mutation.
 They are found in between genes and introns.
 They are inconspicuous – they insert one into another – so if a transposon goes
into another – not a great effect on a gene.
 There also seem to be safe havens – areas of the chromosome in which
transposons really don’t move around – usually heterchromatic (highly
condensed) regions –
 Host is able to inactivate to inactivate – often found in heterochromatin – kept off
by methylation, etc.
Transposable elements and genome structure:
 Useful – structural role around centromeres?
 Other host mechanisms related to those used to suppress virus replication.
 Transposable elements can be harnessed by their hosts – they can drive evolution
of the genome – also play structural roles.
 The other thing that transposable elements can do is since you have homologous
sequences – they can allow recombination events and that can make new
combinations of genes or elements of genes.
GENETIC DISSECTION 1 –Reverse Genetics & Functional Dissection:
 Functional dissection of genomes
 Gene expression analysis – reporter genes/in situ- trying to work from phenotype
back to molecular function.
 Reverse Genetics & gene knockouts (e.g RNA) – starting with molecular info
about a gene and you’re trying to work back to functional info.
Reverse Genetics:
 What genes are encoded in a sequence? Bioinformatics & genome annotation –
identifying where in the sequence particular genes are –
 Another tool is to look whether or not they are conserved between organisms –
comparative genomics valuable for getting info.
 What roles do those genes play: when? Where? What kind of protein? How are
they regulated?
 When? – one can find out when/where using molecular techniques – given that
you know its sequence you can design hybrodization probes to find RNA
expressed by that gene - look for population of RNA in a developmental time or a
certain tissue.

Where? – Expression analysis (transcript + protein) immuno blotting experiments
–
 What does the Gene do? – one can use sequence to knock out the function of the
Gene – so that mutational analysis can follow.
 How? – one can do biochemical experiments on the protein that it produces –
(identity of protein, sub-cellular localization, modifications, interactors).
FUNCTIONAL DISSECTION – GENE EXPRESION STUDIES – mRNA
 In situ hybridization – can be used to tell you where on a chromosome a
particular gene is located –
 Perhaps now a more important use for situ hybridization is to do the same
thing on a tissue section or a whole organism if small – and actually see the
cells that are producing the messenger RNA of your favorite gene.
 Use RNA probe complementary to mRNA (“anti-mRNA)
 Label probe with radioactivity or with tag for colourmetric enzyme
dissection.
 Reporter Genes:
 Promoter of genes includes place where RNA will bind and promoter also
includes regulatory regions that drive tissue specific expression of the gene.
 Reporter protein: some protein not normally expressed – something that’s
really easy to detect in the lab –
