Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.

The pheA gene of Corynebacterium glutamicum encoding prephenate dehydratase was isolated from a gene bank constructed in C. glutamicum. The specific activity of prephenate dehydratase was increased six-fold in strains harboring the cloned gene. Genetic and structural evidence is presented which indicates that prephenate dehydratase and chorismate mutase were catalyzed by separate enzymes in this species. The C. glutamicum pheA gene, subcloned in both orientations with respect to the Escherichia coli vector pUC8, was able to complement an E. coli pheA auxotroph. The nucleotide sequence of the C. glutamicum pheA gene predicts a 315-residue protein product with a molecular weight of 33,740. The deduced protein product demonstrated sequence homology to the C-terminal two-thirds of the bifunctional E. coli enzyme chorismate mutase-P-prephenate dehydratase.     

Molecular cloning and tissue-specific expression of the mutator2 gene (mu2) in Drosophila melanogaster.

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2 gene and rescued the mu2 phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.     

Molecular cloning and expression of a second zebrafish aldehyde dehydrogenase 2 gene (aldh2b).

Aldehyde dehydrogenase 2 (ALDH2) is primarily responsible for detoxification of short-chain aldehydes in vivo. Previously it was reported that zebrafish has an aldh2 gene. Here we report the presence of a second aldh2 gene (aldh2b) in zebrafish. Zebrafish aldh2b locates adjacently to aldh2 on Chromosome 5 and the two genes share the same genomic organizations. aldh2b was predicted to encode a protein comprising 516 amino acids. The protein exhibits 95% amino acid identity with zebrafish ALDH2 and more than 76% identity with other vertebrate ALDH2s, respectively. Employing RT-PCR analysis, we demonstrated that both aldh2 and aldh2b mRNAs were present in embryos at cleavage stage (2 hpf: hour post fertilization) throughout protruding-mouth stage (72 hpf) and in different adult tissues of zebrafish. Taken together, our results reveal that zebrafish has two orthologues of aldh2 gene and the two genes share similar expression patterns during early development and in adult tissues.     

Molecular cloning and nucleotide sequence of leukocidin F-component gene (lukF) from methicillin resistant Staphylococcus aureus.

A lukF gene encoding F-component of Staphylococcal leukocidin from methicillin resistant Staphylococcus aureus (MRSA) was cloned. The nucleotide sequence of lukF gene was determined. The sequence data have revealed an open reading frame, which encodes a polypeptide with 323 amino acid residues. Inspection of the amino acid sequence deduced from nucleotide sequence of lukF and that from F-component of leukocidin from S. aureus V8 clarified that pre-matured F-component contains a typical signal peptide at the NH2 terminus and ATG starting codon for pre-matured F-component was present one base downstream to the TGA which is translation termination codon for S-component of leukocidin [A. Rahman et al. (1991) Biochem. Biophys. Res. Commun. 181, 138-144]. The nucleotide sequence of 5'-flanking region of lukF showed the presence of the consensus sequence of ribosome binding site in the internal region of the structural gene of S-component. The lukF was transcribed in the same direction as that of lukS. No Pribnow box can be discerned in the intercistronic region between the lukS and lukF genes. The amino acid sequence homology between S- and F-components was 31%. F-component was expressed in Escherichia coli DH5 alpha harboring plasmid pFRK92 which contained lukF gene.     

Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene

Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5′ and 3′ remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.     

Lysine excretion by S-(2-aminoethyl)L-cysteine resistant mutants of Bacillus subtilis

S-(2-Aminoethyl)L-cysteine (AEC) at 2 X 10(-1) mM concentration completely inhibited the growth of Bacillus subtilis. This inhibitory effect was readily reversed by 2 X 10(-2) mM L-lysine. Besides L-lysine, L-aspartic acid was only effective of all the natural amino acids tested in reversing the AEC-mediated growth inhibition. AEC resistant mutants of B. subtilis were isolated and found to excrete L-lysine in high yields.     

Molecular cloning and characterization of the Caenorhabditis elegans elongation factor 2 gene (eft-2)

A Caenorhabditis elegans lambda ZAP cDNA library was screened using a fragment amplified from highly conserved regions of the mammalian and Drosophila elongation factor 2 (EF-2). Two types of cDNA clones were obtained, corresponding to two mRNA species with 3'-untranslated regions of 60 and 115 nucleotides, both encoding identical polypeptides. Sequence analysis of these clones and comparisons with hamster and Drosophila EF-2 sequences suggests that they encode C. elegans EF-2. Clone pCef6A, encoding the entire C. elegans EF-2 mRNA sequence including 45 nucleotides of 5'-untranslated region, contains a 2,556-bp open reading frame which predicts a polypeptide of 852 amino acid residues (Mr 94,564). The deduced amino acid sequence is greater than 80% identical to that of mammalian and Drosophila EF-2. Conserved sequence segments shared among a variety of GTP-binding proteins are found in the amino-terminal region. The carboxy-terminal half contains segments unique to EF-2 and its prokaryotic homolog, EF-G, as well as the histidyl residue which is ADP-ribosylated by diphtheria toxin. The C. elegans protein contains a 12-amino-acid insertion between positions 90 and 100, and a 13-amino-acid deletion between positions 237 and 260, relative to hamster EF-2. Partial sequencing of a genomic clone encoding the entire C. elegans EF-2 gene (named eft-2) has so far revealed two introns of 48 and 44 bp following codons Gln-191 and Gln-250, respectively. Southern and Northern blot analyses indicate that eft-2 is a single-copy gene and encodes a 3-kb mRNA species which is present throughout nematode development.     

Molecular cloning,structural analysis,and expression of carp ZP2 gene

The cDNAs encoding carp ZP2 homologous to winter flounder and mammalian ZP2 were cloned. Carp ZP2 contains a tandemly repetitive domain and a nonrepetitive domain. A repeat is composed of 13 amino-acid residues whose consensus sequence is QQTSQQFQPQKPA/V. The length of the repetitive domain is highly variable, but that of the nonrepetitive domain is fairly constant among various cDNAs. The termination codons of various cDNAs appear at three different positions. Three groups of cDNAs were therefore categorized. Groups I–III encode a nonrepetitive domain of 356, 255, and 10 residues, respectively. A carp ZP2 gene corresponding to group II cDNA was cloned. It spans 2.4 kb and consists of eight exons and seven introns. Carp ZP2 mRNA was detected only in oocytes but not in other tissues. Carp ZP2 is heterogenous in size. The molecular weight ranges from 40–80 kDa. It is present in vitellogenic but not in previtellogenic oocytes, nor in other tissues. Carp ZP2 content in oocytes increases as vitellogenesis proceeds. Mol. Reprod. Dev. 46:258–267, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2)

Summary The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line.     

Isolation of a recessive barley mutant resistant to S-(2-aminoethyl)L-cysteine

Summary S-(2-aminoethyl)L-cysteine (AEC) inhibits the growth of mature barley (Hordeum vulgare L vars. Bomi and Maris Mink) embryos grown on sterile medium. This inhibition is relieved by lysine and, to a lesser extent, arginine and ornithine. In order to try and select plants which accumulate lysine, 8200 M2 embryos of sodium azide mutagenised barley were screened for growth in the presence of 0.25 mM AEC. One line, R906 was selected for further characterisation. Progeny of the originally selected plant after selfing were all resistant to AEC. In a reciprocal cross with a sensitive barley the resistant trait was inherited as a single recessive nuclear gene which we designate aec-1.Abbreviation AEC S-(2-aminoethyl)L-cysteine     

Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a

Background  

Nucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression.     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

Molecular cloning and embryonic expression of the Xenopus Arnt gene

Versican, a proteoglycan recently implicated in hair follicle induction, has been shown to influence axon outgrowth in vitro and in vivo. We used immunohistochemistry to study the relationship between versican expression and innervation, during rat vibrissa follicle development and the adult hair cycle. During development, nerve fibres were commonly associated with areas of weak versican expression, and the path of axons appeared to be delineated by sharp boundaries of versican expression. Versican expression changed in the lower follicle dermis during the adult hair follicle cycle but remained strong around the follicle neck reflecting the constant innervation. Our observations show a correlation between versican expression and peripheral innervation indicating that versican may have a dual role in hair follicle biology.