Unformatted text preview: For S. America, they've gotten rid of it all except Bolivia. People didnt' know there were sexual phase. They were clonal, diploid. Later they started using classical genetics. They got evidence there was gene recombinance in the tsetse fly. It's kind of controversial, not really, but hard to understand. A paper came out, showing sexual phase in T. cruzi in the amastigote stage. A new paper came out in Leishmania. It's not general, usually it goes on clonally. She used two markers. T. Bruci is proably the most ussed organism because we know the genome for molecular biology. You can do RNA interference for any gene we want. We can downregulate by adding tetracycline on/off system. That's a nice handle. You can do transformation at a high frequency. Youc an transfect them with plasmids. We have 5-6 drug resistance markers. There's the neo, hygro, flaomycin, etc. You can transfect by using electroporation, using cuvette and electic shock. The membrane gets weak and the DNA get in. There's a high rate of hoomologous recombination so the plasmid's got to have a homologous region. You can destory a function of a gene, or a single site or two sites and replace a region. Gibson took instead of drug resistance, she used florurescent markers with GFP or RFP (red) and then passed them through the tsetse flies. They're not even allowed in the united states. It changes to metacyclics in the salivary glands. Injected these into mice and then cloned them via agar colony or dilution until you get a single cell. She expanded the clones in mice and hibrid them. Reminder for insect, in the human the long slender is the dividing one, and then the short stumpy one which is infective (we can call it metacyclic) that is infective for the insect. They go to the midgut and differentiate in 24 hours to procyclic and lose their surface coat and get a new cyclic coat and then they migrate to hypopharynx and differentiate into ___- which is another stage. What happens when you mix red and green? You get yellow. There's a lot of green ones and red ones. Now, 27 days, she starts to see yellow cells by expressing both red and green via gene recombination. it was the short eppimastigotes that colonize the salivary glands and make. WE don't know what the reduction division that must occur. We don't know what the morphological difference looks like. She's got it down to one cell form which was the epimastigote. Let's switch to transcription. They don't seem to have transcriptional regulation like other cells. They regulate post-transcription and that's how it goes to differentiation. It seems very wasteful but it has been selected for. Maybe this allows them to go through their cuycle very quickly. They found a big surprise. They found 50 genes on the Watson strand 29 genes on the Crick strand. There's a switch point. Every chromosome they sequenced (12) and mini chromosomes and everyone seemed to have these long lines of dgenes. They didn't have operons, and they were just unrelated. It looks like there's a single transcription and it transcribes all these genes together and gets this polycistronic strand. it seems to be RNA polII. Well, they thought ith as to be at the switch, but there's no evidence of a site for a promoter for pol 2. when cells make these giant transcripts yo0u can't use them so they have to break it down into genes. It occursi n C. eleangs , etc called trans-splicing. Every mRNA are the size of the genes nd there are few introns. Only a few genes have introns. Every mRNA had a 39? sequence at the beginning that was the same. Splice leader transcription. 50 to 100 randomly coded genes. They code for an exon and an intron that is removed during transsplicing. He wants to know what the promoter is. It's a polII promoter but it doesn't look like that switch reason. you get 140nt transcript and then this undergoes transsplicing with the mRNA and breaks the polycistron and there's a cleavage polyadenylation and the front is spliced on. Why? They can get constitutive transcription of a large number of genes. Many exons have the cap which is needed for translation. how does this occur. Cis-splicing, there's a lariat structure with A and the two exons are joined. You remove the intron. Trans-splcign, SL RNA and a premRNA. the SLRNA links onto that A and hten another attack linking this to the other exon. It's called trans-esterification. It looks very similar, which one came first. Transsplicing evolved first. Summarize RNApII is used however, one of the first RNAs to be studied... here's that polycistronic here's the switch, mainly from drugresistance studies. There are a bunch of genes called the variant surface gene that causes this... they didn't se........ Gunzl: People had evidence that polI was the one, but this guy proved it. He used epitope tag. There's only one copy of RNA POLI. First he took a phleomycin resistance and it had some homology. He put that in and it underwent double recombination. Now you have a Phleomycin in place of one of those genes. He took another plasmid that had an epitope (something recognized by an Ab). Protein C is a good binder to Ab. He did a transformation without destroying PolI and fused the two together. You'd get 1 molecule of RNAPOLI and a epitope at the end. He could do things like ... here's proof that he has that. It only lit up the band in that mutant. Now you can do immunoprecipitation and deplete and then add protein A sephoarase and you get a supernatant that lacks polymerase I. then he tested it by invitro transcription. He can take lysate that can prove... he incubated with rNTPs, plasmid and target gene. He extracts RNA and assays it by primer extension. You take a oligonucleotide that is complementary to the gene you're interested in. you want to find out how much of that RNA is in the cell. If you take an oligonucleotide, you can put a P32 on it. You can then use reverse transcriptase to extend to the 5' end. Then you do a gel that looks for that piece. You know the exact size the gel should be. This shows he was able to use PolI... It is calcium dependent. ...Here's another protein for loading control. Here's the minus calicium.. Looks like he's removed the tagged RNA PolI. Now there's an extract that is transcriptionally active. Now he can look at what is transcribed and what is not. each band is specific for each gene because we know the size. We have procyclin mRNA. SL is unaffected by depletion of RNA POL I. Which shows that it is RNA POL II. It's definitive proof that PolI transcribes VSG. VSG - variant surface glycoproteins. There's a whole set of microbutubules that go around the cell. There's a coat of 12-25 nm thick. it's the same glycoprotein.... He found this on every trypanosome. It covers the bloodstream form and prevents Abs from penentrating. The only thing blood sees is VSG. They occur as dimers. We know the structure but we don't know the C or N terminus. it is attacedl... GPI anchors, a lipid, glycoprotein. It penetrates the membrane, it's hydrophobic. Then there's the protein. What the antibody sees is the VSG and then at the bottom is the sugar (mannose) monosacchariade. Here's inositol, glocsamine, glycan, ethanolamine, and protein. When the long slender changes to short stumpy, as soon as... you get the l.... and it coats the cell. they don't see a bacteria,m they see.... African traypanosomes - rabbits. Looking at antibodies found in the host (rabbit). The number went up or down before the animals die. It's called relapsing parasetemia. They found Ab sets at each of the the hills. Each paraiste is expressing a new. Each successive wave has a new GS program. lotl. The expression of the VSG occcurs near the telomeres. For a vsg to be espressd,... They have gained aas many .... Heres he VSG by itsel. All these genes are ... firstm but only. Here are the struc. Using the tring. Metacycic 70bp barren region, no genes, what's the process There are two basic m..wtch. Homoolog In-situ They use ES1 and ES... Borst you have VSG arrays and it turns out they're subtelomeric and there's 1200-1400 of those and then you have telomeric VSgs expression sites and other telomeric VSGs that are not at expression sites but in the telomeres. You have gene conversion (left), exchange (middle), and the last one is switching (right). Probably gene conversion is one of the major ones. When they sequenced the gene of T. Bruci, they thought they would find VSGs in the genome, but instead they found pseudogenes and the reversed in sense. These are nonfunctional genes. That led to a whole new theory and proposed by a guy in Glasgow, it didn't fit the paradign that the VSGs are actually mosaics and all using crossing over. The new VSG expression site actually comes from different pieces coming together. it gives itself a much better repertoire of antigens. Instead of 1000s of VSGs but an infinite number with a different antigenicity. That could be the reason this evolved. Remember, there's 100s of minc ...
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- Fall '08
- DNA, new VSG expression, rna poli, randomly coded genes, RNA POL I.