2009Test4 - Winter 2009 Gene Action Exam 4 (Traxler/Gray)...

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Winter 2009 Name: Gene Action Exam 4 (Traxler/Gray) This exam is returned for student use only: not to be photocopied for general distribution. Question 1 (12 points) Recall that OmpF is one of the major outer membrane porins in E. coli . You isolate a strain with an ompF-lacZ translational fusion: in an OmpR + EnvZ + strain, this fusion is toxic to the cell. OmpR- strains expressing this fusion are healthy and grow well at all temperatures, but are LacZ . Part A. Starting with the ompR ompF-lacZ translational fusion strain, describe how you would isolate a mutation in a gene coding for a component of the cellular apparatus for secretion of outer membrane proteins. What additional test would you do to show that your mutation was in the Sec apparatus? ( 75 words.) Part B. What sort of mutation would you expect to recover ( e. g. , nonsense, Tn disruption, deletion, missense, etc.) and why? ( 25 words)
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(14 points) You isolate a strain of Salmonella (which does not have any of its own lac genes) that grows well on peptone. Peptone is a complex mixture of peptides (20-40 amino acids in length, 2000-3000 Da) made by acid hydrolysis of proteins. You characterize a Tn5 transposon mutant library of your strain and discover that mutants that no longer grow on peptone (Pep ) map to two different operons, which contain genes that you call pepA pepB pepC and pepR with the ( σ 70 -dependent) promoters and gene arrangement shown below. p pepABC p pepR pepA pepB pepC pepR Biochemical experiments with cell extracts show that PepC is a peptidase that degrades the larger peptides down to three amino acid peptides in the cytoplasm of the cell. (These tripeptides are subsequently converted to single amino acids by other nonspecific peptidases in the cell .) PepA is an outer membrane porin. PepB is a cytoplasmic membrane peptide permease. Transcription from the pepABC promoter does not occur in the absence of peptone. Additionally, a Tn5 insertion in any one of the 4 pep genes prevents transcription from the pepABC promoter. Part A: You start with uv-mutagenized cells that contain a lacZ transcriptional fusion to the pepABC promoter. How would you identify a mutation that allows constitutive expression of the pepABC operon,? Is your strategy a selection or a screen? ( 60 words) Part B : Which gene do you expect your constitutive mutation to be in? Will your mutation be dominant or recessive? Explain why ( 25 words). Part C
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2009Test4 - Winter 2009 Gene Action Exam 4 (Traxler/Gray)...

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