Scribe.Lecture 18.docx

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Department
Cell and Systems Biology
Course
CSB328H1
Professor
William Navarre
Semester
Winter

Description
Lecture 18: 2 1. bacteria that is already working well and responding to envmt 2. but HGT occurs and new genes are coming in 3. and the new gene coming into the cell must be compatable w the pre-existing operating system 4. how can we explain the GC content of HG acquired islands diff from the host – most often is more AT rich in e coli and salmonella 3 5. first genocmic experiment (analysis of bacteria by chargaff 6. he was the first to discover that bactier have their own unique GC content 7. he finds that serosia and bac has higher GC content 8. homophiles influenza had AT rich 9. also, found that ratios of A to T and G to C approximates to 1 1. A going up, T will go up, C down & G down 4 5 10. HGT has helped bacteria evolve rapidly, rather than having to invent their own antibiotic resistance gene, can acquire it very fast 11. but most HT events decrease fitness 1. new gene that is misregulated will chew through things that shouldnt be chewed, and use resources that depletes resources (ATP) 2. wildtype wil survive better 12. needs to have flexible genome, but also needs to not compromise the preexisting stock that is already there 6 7 13. cells do a lot to block out foreign DNA and try to control it 14. bacteria have unique GC contents and they use it for self-non-self recognition 1. recognizes that diff GC content DNA will be foreign 2. recognition will allow the bacteria to respond 8 15. HNS (heat stable nucleoid structure) is a DNA bidngn protein that….. 16. ….. 1. can bind any DNA 17. very poor DNA binding protein as it exists as a dimer in a cell but exhibitrs cooperative binding behcabiou 9 10 18. hypothesis that HNS is part of the immune system of the cell 1. able to tell inc oming DNA is more AT rich than self 2. HNS polymerizes on the AT rich foreign DNA and silances it and shuts it down 1. this gives the cell the ability to take in DNA but buffers any negative fitness consequences 19. if you get rid of HNS, the AT rich will increase dramatically 20. microarray study of salmonella: comparing the levels of RNA in diff genes 1. genomic islands (grey boxes) are horizontally acquired DNA 2. amount of fold repression by HNS 3. HNS has strong affect at salmonella pathogenicity islands that are AT rich 12 21. each circle is a gene in salmonella genome 22. plotted as GC rich or AT rich (y axis) 23. bulk of genes in slamonella re in the middle 24. big skew of AT rich genes that are repressed 13 25. the avg GC content of an ORF was significantly lower han that of the host genome 26. may not be direct bc we are looking at RNA – HNS can be silencing just 1 TF, and that TF can be repressor which can repress everything else 27. how to show whether HNS is actually binding on the chromosome 14 28. antiody against protein you are looking at, HNS 29. take cells and cross link them with formaldehyde which will cross link protein to DNA, and then you shear DNA and bust the cells open in buffer highe intenstity sound waves 30. and add antibody to it that recognizes HNS, then beads that will pull down certain antibodes which are coated w protein A of staphylococcus aureus (cell wall compoentnst of LPS) that binds the antibodies 31. spinout the beads and the supernatant pellets contain DNA bound by HNS 32. incubate the DNA and then fluorescently label DNA and put it on microarray slide and the fluorescing genes correspond to the genes that were boundto H-NS 15 33. if you now look at the salmonella genome, plotted for its GC content, every spikeis a spike in the AT richness in the genome (vast majority acquired from HGT) 34. H-NS binding (pink) 35. every place where tere is a AT rich, H-NS binds (including the pathogeneicty islands) 17 36. AT rich helicobacter pylori DNA put in GC neural region of the chromosome, and according to the chromatin immuniprecipitation assay, it was being bound aobut 100 fold more by HNS than neighbouring gene (0.16) (proV is a positive control gene – gene that is already silenced by HNS) 37. expression levels showed that it was being silenced 15 fold by HNS 38. gene that slamonella has never seen before but has a protein that polymerizes it bc it cantell it isn’t self dna bc gc content of that DNA is not same as slef dna 18 39. how does this actually occur? AT rich DNA has diff properties than GC rich DNA 19 40. 1. A-tracts: consecutive AATT sequwnce results in narrowing of the minor groove of the DNA and increase in electronegativity here (negatively char
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