Replication origin of the Escherichia coli K-12 chromosome: The size and structure of the minimum DNA segment carrying the information for autonomous replication

Summary A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA ⁺ cells, whereas pBR322 replicates only in polA ⁺ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori⁺ and Ori^- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori^- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Identification of novel genes expressed highly and selectively in the corpora allata of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Applied Entomology and Zoology - Juvenile hormone (JH) has crucial roles in insect physiology, including development, reproduction, and polyphenism. JH is synthesized in the corpora allata (CA)...     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Effects of Chemical Agents on Radiation Inactivation of Streptomyces Protease in Aqueous System

Inactivation of crystalline enzyme, Streptomyces protease G, by γ-ray irradiation in an aqueous system has been investigated. It is indicated that inactivation of the enzyme is attributable mainly to the indirect action of radiation. The inactivation curve is exponential and the G-value for enzyme inactivation is calculated as 0.1 at an enzyme concentration of 1×10^?5m, which is not influenced by varying pH. Effects of various other solutes on radiation inactivation have been also studied. Halogen ions, especially iodine ion, and nitrite ion are most protective among various inorganic anions examined, and alkali metal and alkali earth metal cations are ineffective. Among various organic compounds examined, sulfur-containg compounds and unsaturated compounds are generally effective for protection of enzyme activity against radiation damages. The protective effect of benzene is enhanced by the substitution of electron donating groups. Chloroform and chloral are found to act as a synergist for irradiation inactivation.     

Aroma Constituents of Roasted Coconut

The volatile components of roasted and unroasted dried coconut shreds, isolated by steam distillation, were analyzed by GC and GC-MS. Seventeen compounds were identified from the unroasted coconut, and nine of them were newly identified in coconut meat aroma. Saturated delta-C₈, C₁₀, and C₁₂ lactones were determined as the main components giving the characteristic mild, sweet, and pleasant coconut flavor. The roasted coconut gave the strong characteristic sweet and nutty aroma, and the GC-MS indicated the saturated delta-lactones as main components, and six pyrazines, two furans, and two pyrroles also found seemed to contribute greatly to the nut-like aroma of roasted coconut. The defatted and roasted meal gave a strong nut-like and burnt odor, but not the characteristic sweet aroma. A large increase in pyrazines and other Maillard reaction products and an absence of lactones and fatty acid esters were observed in the volatiles of the roasted-defatted coconut meal.     

Modification of Combined Effect of Radiation and Sodium Chloride on Microorganisms by Chemical Agents during Irradiation

Effects of various chemical agents on the synergistic action of NaCl to the radiation inactivation of bacteria and yeast were studied. The remarkable modification of the radiation lethal effect by some reagents is considered to be a strong evidence for an indirect nature of NaCl synergistic action during irradiation. Most of these modification effects were restricted to the actions during irradiation, supporting the free radical hypothesis in which the short-life active species formed by radiation were considered to attack bacterial cells. Furthermore, pre-irradiation effects under various conditions suggest that the enhancement of radiation lethal effect by NaCl may involve the intracellular events.     

Effect of Irradiation on Microorganisms in a Nuclear Reactor

The effects of irradiation in the JRR-1 (Japan Research Reactor No. 1, a homogeneous light water nuclear reactor; max. power, 50 KW) on microorganisms such as bacterial and fungal spores and yeast cells were investigated in comparison with those of ⁶⁰Co gamma radiation. As far as the lethal effect was concerned the dose rate of radiation in the experimental hole No. 16 of the JRR-1 was equivalent to 3.0×l0⁶~3.4×l0⁶ r/hr with ⁶⁰Co gamma radiation, and a ratio of the neutron effect to the gamma radiation effect on microorganisms in this hole was estimated to be approximately 3~5.4. The results different from those with gamma radiation were obtained in experiments such as post-NaCl treatment and spore germination. The considerable contribution of fast neutrons to the total biological effect of neutrons, in comparison with the thermal neutron effect, could be presumed from the microbiological experiments with the help of physical and chemical data. Morphological changes in post-irradiation growth were observed by means of phase contrast microscopy. No specific aftereffect was found.