gene identification troubles

[R.D. Haigh]

Hi Tammy
I'll present my background first - I'm a bacteria man so some of my 
info will be biased that way - I never did zoology either what sort 
of bug(?) is L.littorea ?

My answers

1.I can't help you here other than to agree it seeems odd you should 
be picking up RNA genes in this sort of screen.

2. The age old problem - you're enormously overjoyed that they're novel 
but what the hell is it anbd no clues available. The obvious next step has 
got to be trying to make knockouts - if these genes are only required for 
stres responses you might expect mutants would be non-lethal in 
permissive conditions, though probably showing phenotypes which might 
help with working out  function. This all requires knockout 
strategies which may not exist for L.littorea (or you may not have 
them) so the next best option is overexpress your gene product, make antibodies and 
check the expression pattern either in whole organisms or cell 
fractions.
By my calculations you are seeing 25-40% (?!*) of your clones are 
coming up with homologs - how good are these? do any of them make any 
sense ?

3. I haven't seen any (but can't say I've looked hard) -  generally 
everything goes into Genbank and if it doesn't, it is probably because 
someone doesn't want it available so you won't find it anyway

4. Sorry not my subject

5. Protein translations are usually very useful - when looking across 
species (especially in  bacteria) DNA matches are usually much poorer 
than protein. I tend to favour blast searches using a translation 
either against the protein database or even a translated version of 
the entire genbank DNA database - all available on the ncbi blast pages
Have you tried any prediction programs - you should be 
able to get a fair idea of the cellular location of your protein but 
do note they are not infallible - PSORT at psort.nibb.ac.jp is 
very good for bacteria but i can't vouch for eukaryotes. there are also 
lots of useful programs (and some crap ones - you'll have to work out 
for yourself) at expasy.hcuge.ch/www/tools.html.

I hope my ramblings are some help

Richard

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    Richard Haigh                      
    Department of Microbiology and Immunology,                        
    University of Leicester,
    University Road,        
    Leicester. LE1 9HN UK
  
    Phone: 0116 2523017 
    FAX   : 0116 2525030 
    Email : rxh at nospam.le.ac.uk
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