HELP!! (: promoter BUT ): NO 5'utr!!!!!

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Thu Apr 13 18:05:17 EST 1995


Subject: DESPERATELY SEEKING ADVICE!

I am back to bug you briefly again... 
I have been very exited about my recent promoter findings: my TATA box 
and CAAT box are a happy 55bp apart, with nearby Sp1 sites, TFIIIA sites, 
NFkB sites, and other fun motifs (It seems too nice for this merely to 
be a coincidental artifact). In addition, I have a 3.9kb clone downstream 
that contains most of the exons from the cDNA clone (preliminary sequence 
data has given me 3 nice exons, beginning with the ATG translational 
start site and ending with part of the  3'untranslated region). The 
exon-intron boundaries are distinct.

Here is the problem: the last match to the cDNA is the ATG translational 
start site: ie/ I cannot find the known 5' untranslated region (144bp) in 
ANY of my clones (I at first thought there was an error in my map, so I 
checked everything).
Just so you know, here is the general map (there is an unaccounted for 
0.7 kb fragment INBETWEEN my supposed promoter element and ATG. I was hoping 
that this would contain my 5'utr, but it did not...)

]--5'--------------------I---------I-----I----------------I----------...3'--] 
           6.0kb           1.5kb     0.7       3.9kb             7.0
                         I promoterI  ?  I ATG-in-ex-in-3'utr...I
          
Do you think my promoter is viable? How can I find my 5' utr? I cannot 
make a probe because there are no decent sites to cut out this region. 
Perhaps I could do PCR. Can I start my promoter analysis without knowing 
this? (ie/ luciferase assay) I am stuck!!

Thanks for any advice you can give me. 

Heidi




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