Nucleotide sequence of the Escherichia coli micA gene required for A/G-specific mismatch repair: identity of micA and mutY.

The Escherichia coli methylation-independent repair pathway specific for A/G mismatches has been shown to require the gene product of micA. Extracts prepared from micA mutants do not form an A/G mismatch-specific DNA-protein complex and do not contain an A/G mismatch-specific nicking activity. Moreover, a partially purified protein fraction containing both A/G mismatch-specific nicking and binding activities restores repair activity in micA mutant extracts. The DNA sequence of a 2.3-kb fragment containing the micA gene has been determined. There are two open reading frames (ORF) in this DNA fragment: one ORF encodes a 25.7-kDa protein whose function is still unknown, the other ORF codes for a protein with an Mr of 39,147, but this ORF can be transcribed and the mRNA can be translated to yield a protein with an apparent Mr of 36 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Deletion analysis showed that this 39.1-kDa ORF is the micA gene as judged by the capacity of the encoded protein to restore the A/G mismatch-specific nicking activity of micA mutant extracts. Furthermore, our results suggest that micA is the same gene as the closely mapped mutY, which encodes the A/G mismatch-specific glycosylase.     

Genetic analysis of the APC gene in taiwanese familial adenomatous polyposis

Colorectal cancer has become the third leading cause of death from cancer in Taiwan. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of multiple adenomatous polyps in the colon and rectum. The gene responsible for FAP(APC) was cloned in 1991. Extensive analyses of the mutation spectra in FAP kindreds have been performed in different countries, but the results have been highly variable (30–80%). In this study, we used denaturing high-performance liquid chromatography (DHPLC) followed by automatic sequencing in an effort to establish the mutation spectrum of APC from DNA of peripheral blood cells. Among the 6 FAP probands analyzed, mutations were detected in 3 (50%), 2 of which were novel. The first novel mutation was at codon 2166, with a C to T transition, resulting in a stop codon. The second novel mutation was at codon 1971, with a C to G transversion, resulting in an amino acid change from serine to cysteine. The third mutation involved an A insertion in the sequence of -AAAAAA- at codons 1554–1556, which created a downstream stop codon (codon 1558). This study is the first to report mutation analysis in Taiwanese FAP probands.     

Ectopic EBP2 expression enhances cyclin E1 expression and induces chromosome instability in HEK293 stable clones

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.     

Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of α-[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3′ side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.