The uvrD gene of Escherichia coli that controls UV sensitivity and spontaneous mutation frequency has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and supl26 mutations were all suppressed by lysogenization of the mutant cells with lambda uvrD+. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg++, Mn++, and Co++ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD- as well as CorA- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the gamma delta sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.

Bacteriophage lambda↗

PubMed1981

Conditional lethality of Escherichia coli strains carrying dnaE and dnaQ mutations.

A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at 28 degrees C but not at 30 degrees C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. A dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperature-sensitive growth mutations were combined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.

Alleles↗

PubMed1981

Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.

By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.

Bacterial Proteins↗

PubMed1981

Studies on oxygen-insensitive nitrofuran reductase in Escherichia coli B/r.

Oxygen-insensitive nitrofuran reductase in Escherichia coli B/r was clearly resolved by DEAE-cellulose column chromatography into two components, one NADPH-linked, and the other both NADPH- and NADH-linked. It is known that the strain requires resistance to nitrofurazone in two mutational steps. It is known that the the first step mutants had no NADPH-linked component and the second step ones had neither this component nor the NAD(P)-H-linked one. The NADPH- and NADH-linked activities of the latter component were similarly inactivated by heat or urea treatment. In addition, it was found that these activities were significantly inhibited by dicoumarol, an NAD(P)H dehydrogenase inhibitor, to similar extents. These results suggest that the activities of the NAD(P)H-linked component originate from a single enzyme. On the other hand, the NADPH-linked component was less sensitive to heat, urea and dicoumarol.

Cell-Free System↗

PubMed1981

Division of patent ductus arteriosus by "isthmus taping method".

"Isthmus taping method" as the safe and simple technique for division of patent ductus arteriosus (PDA), along with surgical and follow-up results were reported. 175 patients with PDA and without other cardiac anomalies were operated by this technique. Most of the patients were operated by junior surgeons as a first step in cardiac surgery. The left chest was entered through posterolateral thoracotomy in the third or fourth intercostal space. Tapes were placed around the isthmus of aorta and PDA. Intercostal arteries were not divided. PDA was divided in-between Potts' clamps. Pulmonary and aortic stumps of the PDA were closed with two rows of continuous over and over suture. Of the 175, one patient (0.6%) with pulmonary hypertension died of postoperative respiratory failure. No surgical hazards of bleeding were noted. No deaths were noted among 131 patients without pulmonary hypertension. Follow-up results in 174 patients up to 14 years were excellent.

Adolescent↗

PubMed1981

Evaluation of mechanical left ventricular assistance with left atrium to aorta bypass in dogs.

Effects of the left heart bypass (LHB) by the left atrium (LA) to the aorta (AO) shunt on the heart were evaluated by measuring such parameters as tension time index (TTI), diastolic pressure time index (DPTI), DPTI/TTI ratio, coronary blood flow and myocardial oxygen consumption (MVO2) in open-chest dogs. A pneumatically driven bypass pump made of polyurethane was utilized in this study. No significant change of MVO2 was observed with surgical maneuver to connect the LA-AO LHB pump to experimental animals. During synchronous pumping of LHB, TTI decreased by 35%, MVO2 decreased by 56% of the control and DPTI/TTI ratio increased by 79%. However, coronary flow decreased by 35%, presumably due to autoregulation mechanism of the coronary circulatory system. Both synchronous and asynchronous LHB demonstrated the same level of effect in volume unloading. However, significant diastolic augmentation was noted only in the former. LA-AO LHB, which has a sufficient supporting effect and does not damage the ventricular wall, was considered to be very useful for temporary support for the failing heart, especially for postoperative low output state.

Animals↗

PubMed1981

Pathological changes in laying hens inoculated with the JPA-1 strain of egg drop syndrome-1976 virus.

Rhode Island Red laying hens were inoculated with the JPA-1 strain of egg drop syndrome-1976 virus and examined pathologically 1, 3, 5, 7, 10, 14, 21, 28 and 80 days postinoculation (p.i.). Eggs with a discolored or soft shell or no shell were laid over a period from 10 to 24 days p.i. Egg production failed transiently in some affected birds after 13 days p.i. At autopsy, the uterus revealed remarkable edema and swelling of the mucosal folds together with the deposition of whitish exudate in the cavity 14 days p.i. Microscopically, intranuclear inclusion bodies were found in epithelial cells of the uterus, isthmus and vaginal gland region 10 and 14 days p.i. There were also severe degeneration and desquamation of the epithelial cells, atrophy of the uterine glands and remarkable infiltration of heterophils, lymphocytes and plasmacytes accompanied with extensive edema. Twenty-one days after inoculation, lymphoid follicles were formed in the mucosal folds of some part of the oviduct. Electron microscopic examination revealed various amounts of virus particles 68 approximately 80 nm in diameter mainly in the nuclei of the epithelial cells on the mucosa and in the exudate, and also in the cytoplasm of macrophages in the uterus.

Adenoviridae Infections↗

PubMed1981