umuC genes (fwd)

Mozart de Azevedo Marins mdamarin at FOX.CCE.USP.BR
Thu Oct 20 13:28:37 EST 1994


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
 





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