Bacterial Genetics Introduction and Recombination 1 (ppt)

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  • Bacterial Genetics Introduction and Recombination 1 (ppt)

    1. 1. Genetic Manipulation and Bacterial Genetics . M J Larkin http://www.qub.ac.uk/mlpage/courses/level3/page.html
    2. 2. Dr Mike Larkin - Communication <ul><li>MBC Room 108 </li></ul><ul><ul><li>xt 2288 (Belfast 90972288 Diverted to the DKB) </li></ul></ul><ul><li>DKB Questor Centre Room 316 </li></ul><ul><ul><li>3rd Floor Microbiology Laboratory </li></ul></ul><ul><ul><li>xt (9097)4390 or (9097)4388 lab’ or (9033) 5577 office </li></ul></ul><ul><li>Email [email_address] </li></ul><ul><li>Pigeon hole in Biology & Biochemistry office </li></ul><ul><li>ALWAYS LEAVE MESSAGE and HOW TO CONTACT YOU! </li></ul>
    3. 3. Questor Microbiology and Environmental Genomics Lab’ http://www.qub.ac.uk/mlpage/page1/index.html
    4. 4. Course Theme. <ul><li>A detailed understanding of the molecular principles that are the basis for the generation of genetic diversity in bacteria. </li></ul><ul><li>Basic knowledge to enable: </li></ul><ul><li>The application of genomic technology in microbiology research - assignment </li></ul><ul><li>E.coli systems ML </li></ul><ul><li>Bacteria other than E. coli JQ </li></ul><ul><li>Pathogens SP </li></ul>
    5. 5. Overview of diversity Environmental Influence <ul><li>DNA Replication </li></ul><ul><li>Mutation </li></ul><ul><li>Repair </li></ul>T o C, pH, eH salinity, growth rate, UV radiation, chemical damage <ul><li>Recombination </li></ul><ul><li>Homologous </li></ul><ul><li>Non-homologous </li></ul><ul><li>Rearrangements </li></ul><ul><li>Deletions </li></ul>DNA Transformation Phage Transduction Plasmid Conjugation
    6. 6. E.coli systems and recombination: Determinants of diversity: Overall aims ML <ul><li>Nine/Ten lectures with Key topics. </li></ul><ul><li>Homologous recombination and DNA repair </li></ul><ul><li>Role of methylation and repair. </li></ul><ul><li>Role of Plasmids; control of replication, transfer and stability. </li></ul><ul><li>Illegitimate recombination: transposons and integrons </li></ul><ul><li>Regulation of DNA transposition. </li></ul><ul><li>You should: </li></ul><ul><ul><li>Have a basic grounding for further reading and other systems covered in the course (e.g pathogens). </li></ul></ul><ul><ul><li>Be able to critically read key papers in the area. </li></ul></ul><ul><ul><li>Critically assess the development of ideas to date. </li></ul></ul>
    7. 7. Homologous Recombination <ul><li>Basic mechanisms </li></ul><ul><li>Recombination in conjugation </li></ul><ul><li>The Holliday Model </li></ul><ul><li>Meselson Radding Model </li></ul><ul><li>Recombination in intact replicons </li></ul><ul><li>Enzyme complex involved </li></ul><ul><li>Role of other genes </li></ul><ul><li>Biochemistry of recombination </li></ul>
    8. 8. Basic mechanisms/models <ul><li>Breaking and Joining of DNA </li></ul><ul><ul><li>Strand exchange. RE COMBINATION </li></ul></ul><ul><li>Types. </li></ul><ul><ul><li>Homologous / general </li></ul></ul><ul><ul><li>Non-homologous / illegitimate </li></ul></ul><ul><ul><li>Site specific </li></ul></ul><ul><ul><li>Replicative / transposition </li></ul></ul><ul><li>HOMOLOGOUS RECOMBINATION </li></ul><ul><ul><li>Reciprocal exchange </li></ul></ul><ul><ul><li>Gene conversion / segregation </li></ul></ul><ul><ul><li>replication not required </li></ul></ul>THEORIES Chiasmatic Janssens 1909 Darlington 1930 Copy Choice Belling 1931 Sturtevant 1949 Lederberg 1955
    9. 9. Recombination in bacteria <ul><li>Recombination in Transformation </li></ul><ul><ul><li>Invasion by Single stranded DNA </li></ul></ul><ul><li>Recombination in Transduction/ Transfection </li></ul><ul><ul><li>Invasion by Double stranded DNA ( Mismatch repair ) </li></ul></ul><ul><li>Recombination in conjugation </li></ul><ul><ul><li>Transfer and invasion by Single stranded DNA </li></ul></ul><ul><ul><li>Recombination between intact Double stranded molecules </li></ul></ul>
    10. 10. Recombination in conjugation Single Stranded DNA Transferred Evidence......... e.g.. Mate Hfr WT ( i.e. LacZ + ) X F - lacZ Mutant mRNA Defective  -galactosidase mRNA Functioning  -galactosidase WT - Invasion recA - NO invasion recBCD - Invasion WT LacZ Single stranded DNA Mutated LacZ
    11. 11. Holliday Model BRANCH MIGRATION Chi -intermediate 3’ 3’ 5’ 5’ A T C G 3’ 3’ 5’ 5’ A T C G 3’ 3’ 5’ 5’ T C G A C A T G C A G T A C T G
    12. 12. Holliday Model contd...... OR Endonuclease Nicking Resolve Resolve T G A C A C T G C A G T C A G T
    13. 13. Isomerisation of a 1-strand X-over to produce a 2-strand X-over. OR 3’end 5’end OR
    14. 14. What happens to mismatched pairs after recombination? Gene conversion . DNA Replication Post-meiotic segregation A C A C A T G C A C Excision A T G C Repair A C Mismatch repair
    15. 15. Meselson- Radding Model for Bacterial Recombination 1. Single strand nick 2. Dissociation of ssDNA 3. Assimilation into D-Loop 4. D-Loop Digestion 5. Ligation 6. Branch migration 7. Complete Recombination
    16. 16. Recombination between Replicons. Chi- intermediates. 1975 Valenzuela & Inman (  -Phage) Potter and Dressler (ColE1 Plasmid) ColE1 amplified in E.coli by chloramphenicol. up to 1000 copies. Many recombine by homologous recombination Extract and examine under EM - high magnification Either INTERLOCKING circles OR Recombinational intermediates
    17. 17. Demonstration of Chi- intermediates. Cut ColE1 at single EcoR1 site. Agarose gel and Et Br stain A B All Linear DNA ? OR Some still Joined ? Observe using EM EcoR1 Cuts TWIST Chi
    18. 18. Resolution of Chi-intermediates. Role of Endonucleases 1 2 3 4 Cuts 2 and 4 Cuts 1 and 3 Cuts 1 and 2 or 3 and 4
    19. 19. Development of Recombination Models <ul><li>“ The prudent Scientist keeps his (sic) hypothesis </li></ul><ul><li>simple. He endows it with only enough complexity to </li></ul><ul><li>account for his observations. </li></ul><ul><li>He stands stubbornly by his simple observations; </li></ul><ul><li>resisting the complicating results of other people’s </li></ul><ul><li>experiments, until there is no further doubt of their </li></ul><ul><li>validity. </li></ul><ul><li>Then he retreats a very short way, taking up a new </li></ul><ul><li>position; with only enough added complexity to </li></ul><ul><li>accommodate the unwanted findings. </li></ul><ul><li>There he digs in and prepares for the next attack of the anarchist.” </li></ul><ul><li>D. Stadler (1973) Annual Review of Genetics Vol7 . </li></ul>
    20. 20. The question remains the same! <ul><li>What is the role of homologous recombination in the evolution of microbial genomes? </li></ul><ul><li>1973 – a detailed answer possible – but mechanisms not known and significance of non- homologous recombination uncertain </li></ul><ul><li>2006 – mechanisms better resolved – must now discuss in relation to transposons and other mechanisms. Whole genomes data confuse the picture! </li></ul>

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