cDNA expression library screening

Zhixiang Chen zchen at uidaho.edu
Tue Oct 7 14:01:51 EST 1997


Dear Colleagues:

When we were analyzing the cDNA clones that we recently isolated with
immunoblotting procedure from a ZAP expression cDNA library, we found that
some of these clones contain considerable amount of 5' untranslated
regions.  Sequence analysis indicated that these 5' untranslated regions
contain stop codons in all the three possible frames that are not
suppressed in the host bacteria we used.  Thus, the coding sequences of the
cDNA clones are not in frame with the lacZ ATG start codon and,
therefore,the only way these clones were identified in our screening (which
requires expressed recombinant proteins) was due to translation initiation
from the ATG codon of the cDNA coding sequences, rather from the ATG codon
of the lacZ gene.  I would appreciate learning whether this is a reasonable
explanation.  For those colleagues who have done a lot of expression
screening (e.g. for isolating gene encoding DNA-binding factors using
promoter sequences as probes), I would also appreciate learning whether
these near-full-length, but out-of-frame clones have ever been isolated in
expression screening and, if so, how frequently they occur.  I surpose that
for efficient translation, there must be some ribosome-binding sequence
upstream of the start codon.  Do the 5' untranslated regions of plant mRNA
contain sequences recognized by E. coli ribosomes?  Thanks for any help.

Zhixiang Chen
University of Idaho







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