Sensitivity of Escherichia coli to viral nucleic acid. VI. Capacity of dna mutants and DNA polymerase-less mutants for multiplication of phiA and phiX 174

Summary Seven groups of dna mutants were tested for the capacity to support the growth of A and X174, using a calcium-dependent transfection system. At the restrictive temperature, two groups of mutants, dnaA and dnaF, allowed the viral multiplication. Group B, C, D, E and G mutants were nonpermissive at 43°C to SS¹ DNA as well as to double-stranded RF molecule. Evidence showing the dispensability for the viral growth of DNA polymerase I and recombination function was also presented. Double mutant deficient in DNA polymerase I and II supported the growth of A sufficiently.     

Genetic studies of the ribosomal proteins in Escherichia coli. XI. Mapping of the genes for L21, L27, S15 and S21 by using hybrid bacteria and over-production of these proteins in the merodiploid strains.

Summary Host capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnaM709 and dnaS707 mutants, multiplication of K was not restricted even at 42°C. In dnaM710 cells, however, growth of K was severely affected at 42°C but not at 33°C. Upon infection of K, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of X174 and 3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with , microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.This paper is number 15 in the series entitled Sensivity of Escherichia coli to Viral Nucleic Acid     

Host factor requirements and some properties of ΦXtB

Summary Replication of XtB, a capsid mutant of bacteriophage X174, depends on the host functions directed by the E. coli genes dnaE, dnaF, dnaG, dnaZ, lig and rep. The cellular products of dnaA, dnaB, dnaC(D), dnaI, dnaP, polA, polB and xth genes are, however, dispensable for the viral growth. In these host factor requirements, XtB resembles phages K and St-1, rather than X174. Host ranges of XtB, St-1 and K overlap considerably, and growth temperature of the three phages is somewhat higher than that of X174. Furthermore, XtB is, like K, inactivated by antiserum against St-1. XtB may thus fill an evolutionary gap between the X174 group and the St-1 group.     

A beta-catenin gradient links the clock and wavefront systems in mouse embryo segmentation

Rhythmic production of vertebral precursors, the somites, causes bilateral columns of embryonic segments to form. This process involves a molecular oscillator--the segmentation clock--whose signal is translated into a spatial, periodic pattern by a complex signalling gradient system within the presomitic mesoderm (PSM). In mouse embryos, Wnt signalling has been implicated in both the clock and gradient mechanisms, but how the Wnt pathway can perform these two functions simultaneously remains unclear. Here, we use a yellow fluorescent protein (YFP)-based, real-time imaging system in mouse embryos to demonstrate that clock oscillations are independent of beta-catenin protein levels. In contrast, we show that the Wnt-signalling gradient is established through a nuclear beta-catenin protein gradient in the posterior PSM. This gradient of nuclear beta-catenin defines the size of the oscillatory field and controls key aspects of PSM maturation and segment formation, emphasizing the central role of Wnt signalling in this process.     

Expression of recombinant Streptolysin O and specific antibody production

Streptolysin O (SLO), an oxygen-labile cytolysin, is the cholesterol-binding exotoxin of hemolytic streptococci. Besides microbiological and pathological interests, this cytolysin has been used as a tool for permeabilization of biomembranes. SLO serves as a diagnostic reagent for determination of anti-SLO antibody titer in streptococcal infection. Availability of highly purified SLO, however, has been limited by low yield in streptococcal culture and purification process. Present subcloning of mature-type full-length SLO gene into an expression vector having strictly controllable araBAD promoter enabled efficient production of the cytolysin. Further, anti-SLO antibody with high specificity was obtained by immunizing with purified SLO protein.     

Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation

Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.     

A targeted mutation of Nkd1 impairs mouse spermatogenesis

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.     

Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide.These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli.Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.     

Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain

Cells of "Paenibacillus fukuinensis" D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed beta-1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu(115) and Asp(176), highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.     

Protein transduction as a strategy for evaluating important factors in mammalian sex determination and differentiation

Protein transduction is a powerful tool to deliver biologically active protein into mammalian cells and whole animals. Transduced proteins are folded properly and can mediate their respective functions in their hosts. To examine the feasibility of applying this strategy to study the molecular events of gonadogenesis, we have studied the kinetics of protein transduction and stability of transduced protein in in vitro mouse gonad culture systems using two reporter proteins, TAT-beta-gal and beta-gal fusion proteins with and without the TAT protein transduction domain (PTD) respectively. Our results indicate that the TAT-PTD was critical and essential for protein transduction to cultured fetal gonads. The TAT-beta-gal reporter entered the cells of the gonads and mesonephros efficiently for both sexes at E11.5 to E15.5 stages examined. The delivered protein persisted in the gonads for an extended period after an initial one-hour transduction. The distribution of the reporter was relatively even in gonads and mesonephros at E11.5 stage for both sexes and at later stages in female. The transduced protein was distributed heterogeneously in male gonads after seminiferous tubule differentiation in which the amount of reporter protein was higher outside than inside the tubules. Nevertheless, we surmise that such protein delivery technique should be useful in studies designed to evaluate the sex determining or differentiating functions of various new protein factors identified by advanced differential screening strategies.