Development and characterization of complex DNA fingerprinting probes for the infectious yeast Candida dubliniensis.
(33/10141)
Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast Candida dubliniensis, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of Candida albicans. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the C. dubliniensis genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly C. dubliniensis-specific probe of the three, generated the most complex pattern, was distributed throughout all C. dubliniensis chromosomes, and separated a worldwide collection of 57 C. dubliniensis isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that C. dubliniensis represents a bona fide species. (+info)
Characterization of the recD gene of Neisseria gonorrhoeae MS11 and the effect of recD inactivation on pilin variation and DNA transformation.
(34/10141)
Pilin antigenic variation in Neisseria gonorrhoeae may result following intrachromosomal recombination between homologous pil genes. Despite extensive study, recA is the only previously characterized gene known to be involved in this process. In this study, the gonococcal recD gene, encoding one subunit of the putative RecBCD holoenzyme, was characterized and its role in pilin variation assessed. The complete recD gene of N. gonorrhoeae MS11 was cloned and its nucleotide sequence determined. The gonococcal recD gene complemented a defined Escherichia coli recD mutant, based on plaque formation of bacteriophage lambda and the restoration of ATP-dependent nuclease activity. Inactivation of the gonococcal recD gene had no measurable effect on cell viability or survival following UV exposure, but did decrease the frequency of DNA transformation approximately threefold. The frequency at which non-parental pilin phenotypes were spawned was 12-fold greater in MS11 recD mutants compared with the parental MS11 rec+ strain. Similar results were obtained using recD mutants that were not competent for DNA transformation. Complementation of the MS11 recD mutant with a wild-type recD gene copy restored the frequency of pilin phenotypic variation to approximately wild-type levels. The nucleotide changes at pilE in the recD mutants were confined to the variable regions of the gene and were similar to changes previously attributed to gene conversion. (+info)
The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC 3.5.1.5) to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme.
(35/10141)
Cyanobacteria assigned to the genus Synechococcus are an important component of oligotrophic marine ecosystems, where their growth may be constrained by low availability of fixed nitrogen. Urea appears to be a major nitrogen resource in the sea, but little molecular information exists about its utilization by marine organisms, including Synechococcus. Oligonucleotide primers were used to amplify a conserved fragment of the urease (urea amidohydrolase, EC 3.5.1.5) coding region from cyanobacteria. A 5.7 kbp region of the genome of the unicellular marine cyanobacterium Synechococcus sp. strain WH7805 was then cloned, and genes encoding three urease structural subunits and four urease accessory proteins were sequenced and identified by homology. The WH7805 urease had a predicted subunit composition typical of bacterial ureases, but the organization of the WH7805 urease genes was unique. Biochemical characteristics of the WH7805 urease enzyme were consistent with the predictions of the sequence data. Physiological data and sequence analysis both suggested that the urease operon may be nitrogen-regulated by the ntcA system in WH7805. Inactivation of the large subunit of urease, ureC, prevented WH7805 and Synechococcus WH8102 from growing on urea, demonstrating that the urease genes cloned are essential to the ability of these cyanobacteria to utilize urea as a nitrogen source. (+info)
Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.
(36/10141)
An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b. (+info)
alpha-soluble N-ethylmaleimide-sensitive factor attachment protein is expressed in pancreatic beta cells and functions in insulin but not gamma-aminobutyric acid secretion.
(37/10141)
The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process. (+info)
Identification of a glucose response element in the promoter of the rat glucagon receptor gene.
(38/10141)
We cloned the 5' upstream region of the rat glucagon receptor gene, demonstrating that the 5' noncoding domain of the glucagon receptor mRNA contained two untranslated exons of 131 and 166 nucleotides (nt), respectively, separated by two introns of 0.6 and 3.2 kilobase pairs. We also observed an alternative splicing involving the 166-base pair exon. Cloning of up to 2 kilobase pairs of the newly identified genomic domain and transfection of various constructs driving a reporter gene, in pancreatic islet cell line INS-1, uncovered a strong glucose regulation of the promoter activity of plasmids containing up to nucleotide -868, or more, upstream from the transcriptional start point. This promoter activity displayed threshold-like behavior, with low activity of the promoter below 5 mM glucose, and maximal activation as of 10 mM glucose. This glucose regulation was mapped to a highly palindromic 19-nucleotide region between nt -545 and -527. Indeed, deletion or mutation of this sequence abolished the glucose regulation. This domain contained two palindromic "E-boxes" CACGTG and CAGCTG separated by 3 nt, a feature similar to the "L4 box" found in the pyruvate kinase L gene promoter. This is the first description of a G protein-coupled receptor gene promoter regulated by glucose. (+info)
Cloning and expression of a novel chicken sulfotransferase cDNA regulated by GH.
(39/10141)
We have used mRNA differential display to compare gene expression in normal and GH receptor-deficient dwarf chickens, and report here the characterization of one differentially expressed gene, which shows significant sequence identity to the sulfotransferase gene family. Partial cDNA clones were isolated from a chicken liver cDNA library and an additional sequence was obtained using 5' rapid amplification of cDNA ends. A complete cDNA probe hybridizes to three transcripts (2.4, 2.0 and 1.45 kb) on Northern blots of chicken liver RNA, which differ in the length of the 3' untranslated region. All three transcripts are expressed at higher levels in normal vs dwarf chickens, as expected for a GH-regulated gene. The expression of this sulfotransferase mRNA was also detected in skeletal muscle, but not other tissues. The administration of GH to chickens increased the hepatic expression within 1 h, suggesting this sulfotransferase could be directly regulated by GH. Sulfotransferase activity, using estradiol or corticosterone as substrate, is detected in cells transfected with an expression vector containing the full-length cDNA. The sequence of this sulfotransferase does not show significant similarity with any subfamily of the sulfotransferases and its endogenous substrate is presently unknown. However, we speculate that GH activation of sulfotransferase activity could play a role in reducing concentrations of growth-antagonistic steroid hormones in GH target tissues. These results demonstrate the usefulness of differential display in this model system to identify genes that play a role in mediating GH action. (+info)
Postsynaptic alpha-neurotoxin gene of the spitting cobra, Naja naja sputatrix: structure, organization, and phylogenetic analysis.
(40/10141)
The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene. (+info)