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14 Cards in this Set
- Front
- Back
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Who injected smooth and rough Streptococcus pneumoniae?
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Fred Griffith
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What was injected into mice that seemed to show transformation?
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Heat-killed smooth colony and a non-virulent colony simultaneously
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How was the cause of transformation discovered?
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Purified Polysaccharide/RNA/Protein/DNA + rough bacteria -- injected into mouse
Purified DNA + rough = DEAD Heat killed enzymes (RNAse/Protease/DNAse) = dead/dead/live mice DNA is the transforming material |
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Why is transformation NOT a highly efficiently process?
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DNA is a very large, hydrophilic (charged) molecule -- not easy to cross cell membrane
Some bacteria (some Streptococcus and Haemophilus species) are naturally competent and take up DNA much faster |
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How is DNA uptake and incorporation performed in a lab?
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E. coli used to grow plasmids
CaCl2 leads to water influx and swelling (why do this?) Electroporation -- put bacteria in low ionic strength buff, shock with brief electric current Ballistic -- tiny metal beads coated with DNA fires at cells |
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What is homologous recombination?
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Break and rejoining of DNA over regions of identical or highly similar sequences
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What is site-specific recombination?
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Breaking and rejoining of DNA at specific enzyme-recognized sites (Lambda)
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What is illegitimate recombination?
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Breaking and rejoining by non-homologous DNA strands
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What protein is required for most E. coli recombination pathways?
Why? |
recA
ATPase, catalyzes strand exchange. Acts as a coprotease (seen in induction in Lambda; degradation of Lambda repressor). Cooperative binding of ssDNA -- recA protein has a higher affinity for ssDNA:recA complex than for ssDNA alone Two DNA binding sites recA mutants (recA-) reduce freq of hfr crosses by about 100,000 fold |
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What does RecBCD do?
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A heterotrimer that separates and degrades DNA.
RecB, RecC -- ATP-dependent helicase RecD -- exonuclease |
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What inhibits the exonuclease activity of RecBCD?
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A "chi" site -- GCTGGTGG
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What direction does RecBCD unwind DNA?
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RecBCD unwinds DNA and acts as a 3' --> 5' exonuclease
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What happens to the ssDNA displaced by RecBCD?
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Bound by RecA and ssb.
Invades another strand (branch migration). |
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How would you use a wild type strain of E. coli to correct an auxotrophic mutant (assume it's Phe-)?
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How do you know you've transformed the mutant strain and not simply contaminated your plate with the wild type E. coli?
How do you know any colonies you isolate are a result of transformation and not a reversion mutation? |
