Hi there,
I don't know if my mail reaches the purpose of this group of
discussion but here I go.
I am interested in the identificatiobn of genes
participating in the SOS response of Streptomyces. In atempts to identify
the umuC gene of Streptomyces coelicolor I have used primers based on
a conserved region of the umuC gene of E. coli when aligned with four
other analogous genes (umuC of Salmonella, mucb, samB and impB that occur
in natural plasmids). I got a PCR product of the expected size, took a
long time cloning and sequencing, and found a sequence that presents the
two regions of the primers when looking one of the open reading frames.
Downstream from the first primer region (NDGCVI) I have three aminoacids (ELY)
that occurs inthe umuC of E. coli and Salmonella and the LY occurs in the
other three proteins. Besides that, there are some spread aminoacids,
groups of two that are conserved in all proteins, or in one or two of
them. I have also compared with the REV1 protein of yeast, a mutator
protein thought to be from the umuC family, and also found some groups of
aminoacids that fit. However, between the two primer regions of my
sequence regions there are eight stop codons. I have wondered of a
diferent organization of the gene in Streptomyces or that I have
amplified some thing related but not exactly what I wanted. But I also
thought that maybe, like the E. coli chromosome region where the umuC
falls, this gene may be in a part of the genome of Streptomyces
coelicolor that was subjected to a series of rearrangements, including
the acquisition of my PCR amplified sequence. To complicate further the
story, the overall GC content of the sequence (63%) is lower than expected
for Streptomyces (68-70%). I have checked for contamination and also have
got positive hibridization signals with the sequence for diferent species
of Strepotmyces.
A lot of my friends told me to give up from this, but the sequence
seems quite curious and I don't want to give up, but also would like to
have some coments before proceeding to the final task: to cklone and
sequence the gene out of a genome library.
Please, any coments from people working with PCR., Strepotmyces
or siilar work would be helpfull.
Thanx.
Mozart
MDAMARIN at FOX.CCE.USP.BR
