diaz@aecom.YU.EDU (Dizzy Dan) (05/05/88)
I am interested in generating mutant strains of E. coli in two genes
I've been working with. I don't want to mutagenize and hunt for mutants
(yuck, what a mess!). The method I use should be simple, effective, and
specific to the genes in question. What if I put an antisense code
for the gene(s) on a plasmid and transform. The antisense message would
then prevent translation of the sense message, right? I'd appreciate some
advice, although, as always, Dan Davison may not respond.
--
dn/dx Dept Molecular Biology diaz@aecom.yu.edu
Dan Diaz Albert Grubelstein College of Osteopathy noordewi@speedy.cs.wisc.edu (Mick Noordewier) (05/07/88)
In article <1784@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes: > I am interested in generating mutant strains of E. coli in two genes > I've been working with. I don't want to mutagenize and hunt for mutants > (yuck, what a mess!). The method I use should be simple, effective, and > specific to the genes in question. What if I put an antisense code > for the gene(s) on a plasmid and transform. The antisense message would > then prevent translation of the sense message, right? I'd appreciate some > advice, although, as always, Dan Davison may not respond. > -- > dn/dx Dept Molecular Biology diaz@aecom.yu.edu > Dan Diaz Albert Grubelstein College of Osteopathy If you are looking for a simple, effective method, then anti-sense is not the answer. This issue received considerable attention several years ago, when anti-sense RNA was proposed as a mechanism for natural gene control. Many workers made efforts, but little seems to have come of it. I actually tried it, in a lambda system, to attempt to regulate expression of a lac fusion. I cloned pBR322 p1 promoter to create anti-beta-galactosidase on a tetracycline-resistant plasmid, then transformed a lambda lysogen which was producing a lac fusion protein. I confirmed the presence of anti-sense transcript by northern blotting, and did dot-blots to ascertain that it was present in excess of the sense RNA (I could distinguish between the sense and anti-sense message because my anti-sense transcript was full-length lacZ, whereas the lac fusion contained only the first few amino acids, therefore I could make a DNA probe that only recognized my anti-sense). I am not suggesting that because I failed the entire concept is faulty, but other's experiences seem to be less than unqualified successes as well. Even the reported successes have variable quality. Issues seem to be stability of the anti-sense transcript, as well as its ability to interrupt a closely coupled transcription-translation mechanism. If you want, I can probably come with some literature citations of both pros and cons. If you try it, good luck. I would be interested in hearing of your results. Mick Noordewier noordewi@cs.wisc.edu
sam@murdu.OZ (Sam Ganesan) (05/08/88)
In article <1784@aecom.YU.EDU> diaz@aecom.YU.EDU (Dizzy Dan) writes: >I am interested in generating mutant strains of E. coli in two genes >I've been working with. I don't want to mutagenize and hunt for mutants >(yuck, what a mess!). Hey Dan, Mutagenising and screening for mutants is not that much of a mess if you know what kind of a mutant you are looking for. Which are genes you are working with. I have been generating spontaneous and oligonucleotide directed mutants in the E. coli gene tyrR and it has not been easy I must admit but has not been that bad either. > The method I use should be simple, effective, and >specific to the genes in question. What if I put an antisense code >for the gene(s) on a plasmid and transform. The antisense message would >then prevent translation of the sense message, right? I'd appreciate some >advice, although, as always, Dan Davison may not respond. I realise antisense RNA was touted as the means of regulation a few years ago but it has quite a few problems. If you really need some references send me a note and I probably can dig up some both for and against. Just out of curiosity, which genes are you working with and what kind of mutants do you want? Sam Ganesan ******************************************************************************* E - mail : ACSnet: sam@murdu.mu.oz JANET: sam%murdu.mu.oz@uk.ac.ukc ARPA : sam%murdu.mu.oz.au@uunet.uu.net sam%murdu.mu.oz@uk.ac.ean-relay UUCP : {uunet,pyramid}!munnari!murdu.mu.oz.au!sam Snail : Sam Ganesan, Microbiology Dept,Melbourne University, Parkville, Victoria 3052, Australia. ******************************************************************************