A novel serine/threonine kinase gene,Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. © 1996 Wiley-Liss, Inc.     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

Cloning,sequence and variability analysis of expressed immunoglobulin light chain genes from yellowtail Seriola quinqueradiata

The cDNA clones encoding immunoglobulin (Ig) light (L) chain variable (V) region associated with constant (C) region were isolated from yellowtail (Seriola quinqueradiata) kidney by expressed sequence tag analysis (accession numbers: AB062619-AB062668, AB064322). The sequences of both VL and CL region contain well-conserved cysteine residues important for intra- and inter-domain interaction in mammals. Comparisons of the amino acid sequence of the CL domain with those of other species showed a high degree of similarity, with 88.3%, 59.8%, and 60.6% to those of wolf fish (Anarhichas lupus), rainbow trout IgL I isotype (Oncorhynchus mykiss) and channel catfish G isotype (Ictalurus punctatus), respectively. Multiple sequence alignments of the CL domain with those of higher vertebrates, however, did not readily allow it to be classified as kappa or lambda isotypes. Furthermore, the pI, hydrophobicity and variability of yellowtail VL regions were studied in 65 cDNA clones and the diversity was observed in CDR1, CDR2 and CDR3 regions.     

Enhanced accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase

We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCS(At)) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCS(At) gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCS(At) gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(gamma-Glu-Cys)(n)-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCS(At) gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl(2) activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd(2+)/mg (dry weight) of cells. The expression of the PCS(At) gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd(2+) approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCS(At) gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd(2+) accumulation in nodules 1.5-fold.     

Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d(5)-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW)(-1) h(-1), respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW)(-1) h(-1), respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds.     

Isolation of a Periplasmic Molecular Chaperone-Like Protein of Rhodobacter sphaeroides f. sp. denitrificans That Is Homologous to the Dipeptide Transport Protein DppA of Escherichia coli

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.     

cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) ( EC 2.3.1.153 ) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases ( St-Pierre et al . (1998 ) Plant J. 14, 703–713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

Bimodal life cycle of the burying beetle Nicrophorus quadripunctatus in relation to its summer reproductive diapause

Abstract 1. Under natural conditions in Kyoto, Japan, the reproductive activities of Nicrophorus quadripunctatus Kraatz (Coleoptera: Silphidae) decreased in summer and the species showed a bimodal life cycle.
2. In the laboratory, most adult pairs raised at 20 °C under a LD 12:12 h regime reproduced when provided with a piece of chicken. In adults raised at 20 °C under a LD 16:8 h regime, however, both reproductive behaviour and ovarian development were reduced. It is concluded that these adults entered a reproductive summer diapause.
3. High temperature (25 °C) also suppressed the reproductive behaviour even under a favourable LD 12:12 h regime. In the field, therefore, adults reduce their reproductive activity in summer because of diapause induced by long-day photoperiods and direct inhibition of reproduction by high temperatures.
4. When the temperature was changed from 20 °C to 25 °C immediately after hatching of larvae, they reached the wandering stage in 95% of adult pairs. When the temperature was changed from 20 °C to 25 °C immediately after oviposition, however, no larvae hatched in 85% of pairs. Egg mortality was significantly higher at 25 °C than at 20 and 22.5 °C; no eggs hatched at 27.5 °C. The physiological mechanisms for reducing reproduction probably prevent the beetles from inefficient oviposition in summer.