Methods of Investigating Gene Expression

University:
California State University, Northridge
Course:
BIOL 360 | Genetics
Academic year:

2023
Views:

447

Pages:

4
Author:

customer-9151926

Peter Lund – Methods of Investigating Gene Expression LECTURE 1 Types of Regulation within a cell Transcriptional regulation → Regulation that affects the amount of mRNA made. Post-transcriptional regulation → Regulation that affects the stability of mRNA made or the translatability of the mRNA. Post-translational regulation → Regulation that affects the stability of protein made. Lac Operon Lac repressor • • • • RNA Pol The lacI gene is expressed all the time (“constitutive”) lacI encodes production of lac repressor Without lactose → lac repressor binds operator and prevents transcription of genes on lac operon With lactose → lactose binds lac repressor which changes its conformation and RNAP can bind and transcribe the genes Nature of mutation State of lac operon in absence of any lactose or glucose* Wild type (no mutation) No transcription Deletion of repressor (lacI) Transcription Deletion of lac promoter No transcription Deletion of lac operator Transcription Lac repressor unable to bind operator Transcription Lac repressor unable to bind lactose/IPTG No transcription *IPTG is not metabolised by the lac operon genes trp Operon Contains genes that encode the biosynthesis of tryptophan. - trpR repressor gene encodes repressor - the presence of tryptophan makes the trp repressor bind and halt transcription Nature of mutation State of trp operon in absence of tryptophan Wild type (no mutation) ON Deletion of repressor (trpR) ON Deletion of trp promoter OFF Deletion of trp operator ON TrpR repressor unable to bind operator ON Trp repressor unable to bind tryptophan ON Lecture 2 In vivo → measuring something that has been made by the living cell (mRNA, protein) In vitro → purify components and replicate aspects involved in the expression of mRNA or protein in a test tube under controlled conditions Both of the above approaches should give the same results. IN VIVO APPROACHES Transcriptional Fusion Fusing a promoter of a gene to a gene whose product can easily be assayed. The vectors used for this are often called promoter probes. Measuring mRNA levels directly using Northern Blotting Isolating total mRNA from a cell and detecting how much RNA was present in the cell at that time. The RNA will hybridise to a radioactive probe on a filter and then exposed to X-ray film. Where the band lies will tell you the amount of RNA that was extracted. Northern blots CAN give a total transcript size. Northern blots CANNOT map the start point of the mRNA.