微生物学 美国Indiana University Purdue University授课04.pptVIP

Strategies for StudyingMicrobial Pathogenesis BIOL 533 Lecture 4 Reporter Gene Fusions Operon fusion: transcriptional Promoter from foreign gene Translational sequence from reporter gene Reporter Gene Fusions Gene fusion: translational Promoter and translational signal from foreign gene Initiation signal/+NH2 coding sequence Translational signal also from reporter gene Initiation sequence and part of NH2 sequence of reporter gene missing, but COOH region still present Two coding sequences must be in frame Technique Use transposon vector containing lac fusion Carries promoterless lacZ gene and selectable antibiotic marker Selection for antibiotic marker generates set of random insertions in chromosome Colonies carrying transposon screened for expression under conditions tested Foreign Gene Expression Assay for production of βgal Higher temperature +— iron etc. Locate unknown gene lacZ Fusion Used to find regulatory genes that control expression of virulence gene lacZ fusion strain is mutagenized (chemical or another transposon) Resulting colonies screened for aberrant regulation lacZ Fusion Aberrant regulation: Colonies are not Lac+ in presence of inducing conditions Loss of activator Colonies are Lac+ under both inducing and non-inducing conditions Loss of repressor lacZ Fusion Gene tagged by transposon can be cloned Segment of DNA adjacent to transposon: Can be used as hybridization probe to clone wild-type locus Can also be cloned by complementation Cloned DNA can be sequenced phoA Gene Fusions Virulence genes are small subset of total genes, so random mutagenesis will affect many more non-virulence genes Way to optimize number of virulence gene mutations takes advantage of fact that most virulence genes are on bacterial surface or excreted into medium phoA Gene Fusions Translational protein fusion Alkaline phosphatase is expressed only in periplasm Foreign gene product has to be expressed outside of cytoplasm In vector, signal sequence mutations block ex