The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCIDresponsible gene. We isolated an ORF of the murine DNAPKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.
Escherichia coli mutants sensitive to methyl viologen (MV), an active oxygen propagator, were isolated. Among them, the new genes mvrA and mvrB were mapped at 7 and 28 min on the E. coli linkage map, respectively. MV toxicity was exerted only in the presence of oxygen and was suppressed by the radical scavenger uric acid but not by the hydroxyl radical scavenger mannitol. The mvr mutants were sensitive only to MV and had a normal repair capacity for the MV-damaged DNA. From these results, these mutants were assumed to be related to the elimination of MV-specific toxic species. Gene mvrA was cloned into vector pBR322 and its sequence was determined. The mvrA gene, which was predicted to range in size from 600 to 900 base pairs (bp) by transposon TnlOOO insertion analysis, was identified to be 807 bp, with an approximately 60-bp promoter sequence carrying consensus sequences for the -35 region, the -10 region, and a ribosome-binding site. The MvrA protein deduced from the DNA sequence was 29.7 kilodaltons, which was in good agreement with the 29 kilodaltons of the MvrA protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after a maxicell labeling experiment.Active oxygens are produced in respiring cells and are assumed to be a major cause of oxygen toxicity (13,14). Since Escherichia coli cells are continually exposed to active oxygens during respiration as well as during exposure to ionizing radiation (35), they have defense mechanisms against them. These mechanisms include superoxide dismutase against the superoxide radical (29); peroxidase and catalase against hydrogen peroxide; and antioxidants and radical scavengers like glutathione, ascorbic acid, vitamin E, and uric acid. Nonetheless, a mutant strain of E. coli polA recB was found to be inviable due to the deficient repair of DNA damage produced by oxygen, an indication that DNA repair genes are required for the defense against oxygen toxicity (30). Thus, E. coli seems to have at least two kinds of defense mechanisms against oxygen toxicity: the elimination of active oxygens and the repair of damaged DNA. Since active oxygens are assumed to be a major cause of many important biological phenomena such as mutation, cancer (1, 37), and aging (8), it is of great importance to know the defense mechanisms against them. Methyl viologen (MV), or paraquat, is a powerful superoxide radical propagator (12,17,18). MV is readily reduced by a single electron transfer by NADPH diaphorase (18) or by NADPH cytochrome P-450 reductase (6) to a stable but oxygen-sensitive monocation radical. It rapidly reacts with oxygen, with the generation of truly toxic species, superoxide radical, and subsequently, hydrogen peroxide (12). The secondary reactions between these two species produces a hydroxyl radical (36,39,40). To elucidate the protection mechanisms against oxygen toxicity, mutants of E. coli sensitive to MV were isolated, their properties were examined, and the mutations were mapped. Its relevant gene mvrA was cloned and analyzed. MATERIALS AND M...
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