Identification of repair enzymes for 5-formyluracil in DNA. Nth, Nei, and MutM proteins of Escherichia coli

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth, nei, or mutM gene into the E. coli triple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH(4). Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in the E. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.     

Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA

The pathways leading to G:C→C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C→C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E.coli. By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.coli. The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.coli. Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.coli MutM protein, could specifically bind to the C/C mismatches in DNA.     

Extracellular Formation of O-Butylhomoserine by Anaerobes

Clostridium BB–264, Cl. acetobutyricum 314–48, Cl. kaneboi, Cl. saccharoperbutylacetonicum and other two strains of Cl isolated recently produced an unidentified ninhydrin-positive compound in medium containing 5 % glucose, 1 % ammonium acetate, 0.1 % potassium dihydrogen phosphate, 0.04 % magnesium sulfate, 0.001 % ferrous sulfate, 0.1 % yeast extract, 10 μg/liter of biotin and 1 % calcium carbonate.

This ninhydrin-positive compound was eluted with solvent composed of butanol: acetic acid: water (4: 1: 2) by chromatography on cellulose powder column. It was crystallized from ethanol and then identified as an amino acid, O-butylhomoserine (Abbrev. as O-BHSer). Yield of this amino acid increased by adding homoserine or butanol to the medium. The increase was also recognized with addition of glycine, lysine, serine, threonine or valine. The formation of this amino acid was repressed by adding methionine to the medium.

Gas pressure to the culture is one of the important factors that make the amino acid formation by anaerobes possible.     

Contribution of Zinc Deficiency to Insulin Resistance in Patients with Primary Biliary Cirrhosis

The relationship between metabolic abnormalities of trace elements and insulin resistance has been established. Recent studies have revealed that insulin resistance is associated with autoimmune responses. The purpose of this study was to examine the correlation between zinc or copper metabolism and insulin resistance in patients with primary biliary cirrhosis (PBC). Sixteen patients with PBC were divided into two groups: early and advanced stage disease. The overall value of the homeostasis model assessment of insulin resistance (HOMA-IR) in patients with advanced stage PBC was significantly higher than that in patients with early stage PBC, although the mean value in advanced stage PBC was significantly lower than that in hepatitis C virus (HCV)-related liver cirrhosis. There was an inverse correlation between serum zinc concentrations and HOMA-IR values in patients with PBC, while we found no correlation between serum copper levels and HOMA-IR values. HOMA-IR values were inversely associated with peripheral platelet counts, indicating the relationship between insulin resistance and hepatic fibrosis. These results suggest that zinc deficiency plays important roles of insulin resistance and subsequent hepatic fibrosis in patients with PBC, although insulin resistance in advanced stage PBC was significantly milder than that in HCV-related liver cirrhosis.     

Interferon regulatory factor-2 induces megakaryopoiesis in mouse bone marrow hematopoietic cells

Megakaryopoiesis is associated with inflammatory reactions. To investigate the role of interferon regulatory factors (IRFs) in inflammation-associated megakaryopoiesis, mouse bone marrow hematopoietic stem cells (HSCs) were analyzed. IFN-γ treatment induced IRF-2 expression as well as the expression of CD41 and IRF-1 in HSCs. An in vitro clonogenic assay showed that IRF-2- but not IRF-1-overexpressing cells increased the number of megakaryocytic colonies. IRF-2 transfection up-regulated CD41 promoter activity in hematopoietic cell lines. The number of CD41-positive bone marrow cells increased in mice injected with IRF-2-expressing bone marrow cells. These findings suggest that IRF-2 plays an important role in megakaryopoiesis in inflammatory states.