groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay.

A groEL gene-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus (Bst) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion-binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder-like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species-specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100-fold higher than that for conventional polymerase chain reaction (PCR). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the groEL gene-based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus, and it could be used in field diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The loop-mediated isothermal amplification (LAMP) assay using groEL gene (an abundant, highly conserved gene and member of the groESL chaperone gene family) provided rapid, species-specific and highly sensitive method for detecting Vibrio parahaemolyticus, the leading causal agent of seafood-borne diseases worldwide. Moreover, groEL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism.

Development of a groEL gene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus.

AIMS: To develop an effective multiplex polymerase chain reaction (PCR) for the simultaneous detection of three important Vibrio species, Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) using the groEL gene, a potential phylogenetic marker. METHODS AND RESULTS: Three species-specific primer sets were designed to target Vc, Vp and Vv. A total of 131 Vibrio and non-Vibrio strains were used to determine the specificity and sensitivity of primers. The primers produced specific PCR fragments from all target species strains and did not cross-react with other Vibrio and non-Vibrio species. This PCR method showed good efficiency in detecting coexisting target species in the same sample with a detection limit of 100 pg of Vc, Vp and Vv from mixed purified DNA. Detection of three target species was also possible from artificially inoculated shellfish, flounder and sea water. CONCLUSIONS: The groEL gene is a potential marker for accurate simultaneous detection of Vc, Vp and Vv and could be used to detect these species in environmental and clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This newly developed multiplex PCR is a useful and cost-effective method that is applicable in a disease-outbreak prediction system and may provide an effective tool for both the epidemiologist and ecologist.

Application of groEL gene for the species-specific detection of Vibrio parahaemolyticus by PCR.

AIMS: Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders and is transmitted through ingestion of raw or undercooked contaminated seafood. We used the groEL gene for the species-specific detection of V. parahaemolyticus from artificially inoculated shellfish, fish and seawater. METHODS AND RESULTS: The nucleotide sequences of 24 Vibrio and seven non-Vibrio spp. were compared, and less conserved regions were selected for the designing of primer sets. To detect V. parahaemolyticus specifically, PCR conditions were standardized and tested to evaluate the specificity of primers. A 510-bp band was appeared only from V. parahaemolyticus by PCR. Notably, the detection was shown to be functional at high annealing temperature above 68°C. The groEL primers detected 100 pg and 1 ng of DNA purified from V. parahaemolyticus culture and artificially infected oyster tissue, respectively. CONCLUSIONS: The groEL gene is a potential marker for the species-specific detection of V. parahaemolyticus and could be used to detect this bacterium in contaminated food by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR using primers designed from groEL gene provide an efficient method for the accurate identification of V. parahaemolyticus from contaminated samples.

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction.

Vibrio anguillarum, an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum. PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum. Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA, or from six colony-forming units (CFU) mL(-1) of cultured V. anguillarum. However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g(-1) tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.

Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus.

The structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced. The vmhA gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a predicted molecular mass of 83,059. The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El Tor hemolysin.

Cloning and identification of a phospholipase gene from vibrio mimicus.

The phospholipase gene phl was identified from Vibrio mimicus (ATCC33653) and sequenced. The entire open reading frame (ORF) was composed of 1410 nucleotides and encoding 470 amino acids. The phl was placed upstream of hemolysin gene (vmhA) with opposite direction of transcription. From the BLAST search program, the deduced amino acids sequence showed 74.4% identity with phospholipase gene (lec) from V. cholerae El Tor. The entire ORF of phospholipase gene was amplified by PCR and inserted into an Escherichia coli expression vector, pET22b(+) and introduced E. coli BL21(DE3). SDS-PAGE demonstrated that a protein corresponding to the phospholipase was overexpressed and migrated at a molecular mass of 53 kDa.

Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus.

The vmc gene encoding a metalloprotease of Vibrio mimicus (ATCC 33653) was cloned in Escherichia coli and sequenced. The vmc gene contained 1884 nt sequence which codes a polypeptide of 628 amino acids with a predicted molecular mass of 71,275 Da. The deduced amino acid sequence had the similarity of 68.5% with V. parahaemolyticus metalloprotease. The consensus sequence of a zinc binding motif (HEXXH) was identified to be HEYTH. The zymography analysis showed a gelatinolytic protein band around molecular mass of 61 kDa, and this result suggested that the cloned metalloprotease may undergo processing during secretion.

Characterization of different forms of rat mammary gland acetyl-coenzyme A carboxylase mRNA: analysis of heterogeneity in the 5' end.

Acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) catalyzes the rate-limiting reaction in the biogenesis of long-chain fatty acids. We have previously reported the coding sequence of ACC mRNA from the mammary gland of the lactating rat. The existence, in this tissue, of several forms of ACC mRNA with different 5'-untranslated regions has now been established. Two mRNAs constitute the major ACC mRNA species, they differ from one another in the presence or absence of a 61-nucleotide fragment at the center of the 5'-untranslated region. Multiple forms of ACC mRNA might originate through differential splicing of the primary transcript.

A novel technique for the effective production of short peptide analogs from concatameric short peptide multimers.

We designed a basic unit of the modified chicken gonadotropin releasing hormone II (cGnRH-II) peptide containing a trypsin cleavable linker peptide at both ends of the original peptide. We made a synthetic DNA coding for the modified cGnRH-II peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The expressed peptide multimers were purified and subject to amino-terminal sequence analysis, which displayed the amino acid sequences expected from the designed nucleotides of the expression cassette. The monomeric cGnRH-II peptide analogs were generated after trypsin digestion. The present results showed that the technique developed for the production of the concatameric peptide multimers with cleavable linker peptides can be generally applicable to the production of short peptide analogs.

Comparison of cell proliferation and epigenetic modification of gene expression patterns in canine foetal fibroblasts and adipose tissue-derived mesenchymal stem cells.

OBJECTIVES: This study compared rate of cell proliferation, viability, cell size, expression patterns of genes related to pluripotency and epigenetic modification between canine foetal fibroblasts (cFF) and canine adipose tissue-derived mesenchymal stem cells (cAd-MSC). MATERIALS AND METHODS: Proliferation pattern, cell viability as well as cell size at each passage of cFF and cAd-MSC were measured when cultures reached confluence. In addition, real-time PCR was performed to investigate expression of Dnmt1, HDAC1, OCT4, SOX2, BAX, BCL2 genes with reference to ß-actin gene expression as an endogenous control in both cell lines. RESULTS: cFF and cAd-MSC differed in number of generations, but not in doubling times, at all passages. Mean cell size of cAd-MSC was significantly smaller than that of cFF. Cell viability was significantly lower in cFFs and apoptotic level was significantly lower in cAd-MSC compared to passage-matched cFF. In the expression of genes related to pluripotency and epigenetic modification, level of HDAC1 in cAd-MSC was significantly higher than in cFF, but expression of Dnmt1 did not differ between the two groups. OCT4 and SOX2 were significantly more highly expressed in cAd-MSC compared to cFF. CONCLUSIONS: cAd-MSC have higher stem-cell potential than cFF in terms of proliferation patterns, epigenetic modification and pluripotency, thus cAd-MSC could be more appropriate than cFF as donors of nuclei in somatic cell nuclear transfer for transgenesis.

Acetyl-CoA carboxylase mRNA species with or without inhibitory coding sequence for Ser-1200 phosphorylation.

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in the biogenesis of long chain fatty acids. The phosphorylation of the Ser-1200 residue by cyclic AMP-dependent protein kinase transforms ACC from a citrate-independent form to a citrate-dependent form (10, 16). We have isolated ACC cDNA clones with and without 24 bases which code for 8 additional amino acids located 4 residues upstream to the Ser-1200. The presence of the 8 extra amino acids inhibits the in vitro phosphorylation of the Ser-1200 by the catalytic subunit of cyclic AMP-dependent protein kinase. The S1 nuclease protection experiments indicate that the corresponding two ACC mRNA species occur in vivo. Furthermore, the occurrence of the two forms of ACC mRNA is regulated under different physiological conditions for lipogenesis in a tissue-specific manner. The existence of two forms of ACC mRNA provides the basis for the existence of isozymes of ACC whose Ser-1200 can be selectively phosphorylated. The location of this regulatory sequence for a specific phosphorylation site represents a new regulatory mechanism for protein phosphorylation.

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli.

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.

Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase.

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.2) catalyzes the rate-limiting reaction in long-chain fatty acid biosynthesis. To investigate the mechanism of genetic control of expression of Ac-CoA carboxylase and the relationship between its structure and function, cDNA clones for Ac-CoA carboxylase were isolated. The complete coding sequence contains 7035 bases; it encodes a polypeptide chain of 2345 amino acids having a Mr of 265,220. The sequences of several CNBr peptides of Ac-CoA carboxylase were localized within the predicted protein sequence as were those peptides that contain the sites for phosphorylation. The deduced protein contains one putative site for biotinylation in the NH2-terminal half. The "conserved" biotinylation site peptide, Met-Lys-Met, is preceded by valine, whereas alanine is found in a similar position in all other known biotin-containing proteins. The primary sequences of Ac-CoA carboxylase and carbamoyl phosphate synthetase exhibit substantial identity.

beta-Agarase from Pseudomonas sp. W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity.

The recombinant plasmid (pJAI), harbouring the agarase gene (pjaA) of Pseudomonas sp. W7, was introduced and expressed in Escherichia coli JM83. The agarase was purified using a combination of acetone precipitation and anion-exchange, gel-filtration and affinity chromatographies, with overall yield of 10% from the culture supernatant of E. coli JM83 (pJAI). The purified agarase migrated as a single band (molecular mass 59 kDa) on SDS/PAGE and was found to be beta-agarase, which could hydrolyse the beta-1,4 linkage of agarose to yield neoagarotetraose as the main product. Optimal enzyme activity was at pH 7.8 and the temperature optimum spanned the broad range 20-40 degrees C. The recombinant agarase was halophilic, maximum activity being exhibited at 0.9 M NaCl. This halophilic property could improve the production of neoagaro-oligosaccharides available in a marine environment.

Cloning of human acetyl-CoA carboxylase cDNA.

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. In order to understand the mechanisms that regulate human acetyl-CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human acetyl-CoA-carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85% identity in nucleotide sequence with previously cloned rat acetyl-CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl-CoA-carboxylase mRNA species, which differ in the 5' untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type II acetyl-CoA-carboxylase mRNA contain 313- and 173-base-long 5' untranslated regions, respectively. The first 240 nucleotides in the 5' untranslated region of type I acetyl-CoA-carboxylase mRNA replace the first 100 nucleotides of the (G + C)-rich region of the 5' untranslated region of the type II mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue-specific manner.

RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.