Gene Regulation 2 Flashcards
1. Explain what promoter and enhancer traps are and how they are used 2. Explain how a combination of regulatory steps can be used to control a process in time and space
What are basal promoters?
binds the transcription factors required for recruitment of RNA polymerase, and for transcription initiation
What are enhancer sequences?
regulatory DNA sequences that, when bound by transcription factors, increase the transcription of an associated gene
What are some key features of enhances?
- are position independent
- are orientation independent
- are up to 100kb away but usually within 2-10 kb of gene(s)
- regulate the time, place, and amount of transcription
What prevents enhancers from acting on other promoters that are within their reach?
boundary elements or insulators
What is an enhancer trap?
a reporter gene with a promoter lacking any enhancer elements
What is a promoter trap?
a reporter gene having no promoter
What do both promoter and enhancer traps require?
DNA elements necessary to allow for transformation in the organism of choice
What do enhancer and promoter traps do?
- reporter gene is expressed only when it lies close to the enhancer trap or within the enhancer or promoter trap of another gene
- the expression pattern of the reporter gene in a transformed organism depends on the enhancer elements of the gene near or in which the reporter gene inserts
Why do we use enhancer and promoter traps?
- identify enhancer and promoters that function in different tissues or time of development to use in engineering the expression of other genes
- identify genes transcribed in various spatial or temporal patterns during development
What are the steps in an enhancer/promoter trap experiment?
- Make an appropriate reporter construct for the organism of interest in an E. coli plasmid and isolate plasmid DNA
- Transform organism of interest with the construct such that the construct will insert into random locations on the genome
- Screen progeny of transformants for the expression of the reporter gene to detect the specific pattern
- Clone the gene/promoter/enhancer DNA adjacent to the promoter/enhancer trap
What are p elements?
a transposon that can move from one site in the DNA to another
How are p elements used?
- a nuclease cuts the p element ends to promote this transposition
- replace the transposase gene with a sequence of interest
- transposase will need to be provided for this engineered construct to be inserted into the target genome
- reporter gene will be transcribed only if it is inserted in the genome within the transcribed region of another gene (between promoter and terminator)
What is an enhancer trap?
- Enhancer trap is a reporter gene with a promoter but no enhancers
- The reporter gene will be strongly transcribed only if inserted in the genome near an enhancer element of another gene
Draw a vector, with a p element, basal promoter, and reporter gene
look on pg 3 in lecture 7
or
look on lecture 7 slide 19-20
How can we find the sequence next to the reporter gene?
- Isolate and fragment the genomic DNA with a restriction endonuclease
- Inverse PCR
a. Ligate the ends of the fragments – small circular pieces of DNA generated
b. PCR – amplify the sequences found around the reporter gene - Sequence the PCR fragment and identify the location of this sequence within the genome
