Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6 ⌬1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6 ⌬1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin-and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin-and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin-and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

A study was designed to describe the molecular epidemiology of CTX-M-producing Escherichia coli over a 6-year period (2000 to 2005) in a large well-defined Canadian region with a centralized laboratory system. Molecular characterization was done by isoelectric focusing, PCR, and automated sequencing, while genetic relatedness was determined by pulsed-field gel electrophoresis with XbaI. Of the 552 viable extended-spectrum ␤-lactamase-producing E. coli isolates isolated, 354 (64%) were positive for bla CTX-M genes associated with Members of the family Enterobacteriaceae, especially Klebsiella spp. producing extended-spectrum ␤-lactamases (ESBLs), such as the SHV and TEM types, have been recognized since the 1980s as major causes of hospital-acquired infections (20). These infections were mainly associated with epidemic clones in the intensive care setting. From the late 1990s to the present, Enterobacteriaceae (mostly Escherichia coli) producing different ESBLs, such as the CTX-M enzymes, have emerged within the hospital and community settings as important causes of urinary tract infections (UTIs) (1). The majority of studies that have investigated community-onset infections caused by CTX-M-producing E. coli isolates showed that these isolates are often not clonally related, although clusters have been described in the United Kingdom and Canada (25).The CTX-M ␤-lactamases, of which there are now over 50 different types, have originated from Kluyvera spp. and can be divided into five groups on the basis of their amino acid identities (3): the CTX-M-1 group, the CTX-M-2 group, the CTX-M-8 group, the CTX-M-9 group, and the CTX-M-25 group.CTX-M ␤-lactamases have become the most prevalent type of ESBLs described during the last 5 years, especially from certain European and South American countries (7). CTX-M-producing E. coli isolates isolated from hospital and community sites often exhibited coresistance to trimethoprim-sulfamethoxazole, tetracycline, gentamicin, tobramycin, and ciprofloxacin. The CTX-M enzymes have been associated with the presence of different qnr genes and the production of a novel AAC(6Ј)-Ib aminoglycosidemodifying enzyme that has the additional ability to modify certain fluoroquinolones (7).Very limited data regarding the molecular epidemiology of ESBL-producing Enterobacteriaceae in large geographical areas are available (15). Previous studies in the Calgary Health Region of Canada have shown that E. coli is responsible for more than 95% of the ESBL prevalence in that region and that the majority of isolates are positive for bla CTX-M ␤-lactamases (23). We also noted that the majority of infections originated from the community and that a CTX-M-14-producing cluster was responsible for community outbreaks of UTIs during 2000 and 2001 (21, 23). We designed a study that investigated the molecular epidemiology of CTX-M-producing E. coli isolates isolated in the Calgary Health Region over a 6-year period (January 2000 to December 2005) and determined if these bacteria were introduced into regional ...

Eukaryotic genes encoding ubiquitin-congugating enzyme (Ubc)-like proteins have been isolated from both human and yeast cells. The CROC-1 gene was isolated by its ability to transactivate c- fos expression in cell culture through a tandem repeat enhancer sequence. The yeast MMS2 gene was cloned by its ability to complement the methyl methanesulfonate sensitivity of the mms2-1 mutant and was later shown to be involved in DNA post-replication repair. We report here the identification of a human MMS2 ( hMMS2 ) cDNA encoding a novel human Ubc-like protein. hMMS2 and CROC-1 share >90% amino acid sequence identity, but their DNA probes hybridize to distinct transcripts. hMMS2 and CROC-1 also share approximately 50% identity and 75% similarity with the entire length of yeast Mms2. Unlike CROC-1 , whose transcript appears to be elevated in all tumor cell lines examined, the hMMS2 transcript is only elevated in some tumor cell lines. Collectively, these results indicate that eukaryotic cells may contain a highly conserved family of Ubc-like proteins that play roles in diverse cellular processes, ranging from DNA repair to signal transduction and cell differentiation. The hMMS2 and CROC-1 genes are able to functionally complement the yeast mms2 defects with regard to sensitivity to DNA damaging agents and spontaneous mutagenesis. Conversely, both MMS2 and hMMS2 were able to transactivate a c- fos - CAT reporter gene in Rat-1 cells in a transient co-transfection assay. We propose that either these proteins function in a common cellular process, such as DNA repair, or they exert their diverse biological roles through a similar biochemical interaction relative to ubiquitination.