Molecular marine biology and biotechnology最新文献

Random amplified polymorphic DNA (RAPD) analysis was used to amplify the genome of black tiger prawns (Penaeus monodon) to detect DNA markers and assess the utility of the RAPD method for investigating genetic variation in wild P. monodon in Thailand. A total of 200 ten-base primers were screened, and 84 primers yielded amplification products. Six positive primers that gave highly reproducible RAPD patterns were selected for the analysis of three geographically different samples of Thai P. monodon. A total of 70 reproducible RAPD fragments ranging in size from 200 to 2000 bp were scored, and 40 fragments (57%) were polymorphic. The RAPD analysis of broodstocks from three different locales, Satun-Trang, Trat, and Angsila, revealed different levels of genetic variability among the samples. The percentages of polymorphic bands were 48% and 45% in Satun-Trang and Trat, respectively, suggesting a high genetic variability of the two samples to be used in selective breeding programs. Only 25% polymorphic bands were found in the Angsila sample, indicating the lowest polymorphic level among the three samples examined. Primer 428 detected a RAPD marker that was found only in P. monodon originating from Satun-Trang, suggesting the potential use of this marker as a population-specific marker in this species.

The ability of zebrafish skeletal muscles to internalize and express plasmid DNA was demonstrated using pCMVCAT1, a chloramphenicol acetyltransferase (CAT) construct driven by the human cytomegalovirus immediate early (CMV-IE) promoter. We found that CAT activity was correlated to the amount of plasmid DNA injected, with maximal expression at 5 micrograms of pCMVCAT1. CAT activity was also shown to increase steadily over the first seven days after injection, with high levels of CAT expression persisting up to one year. Intramuscular injection of CAT constructs driven by other viral promoters also resulted in high levels of CAT activity. Histochemical localization using a CMV beta-galactosidase construct confirmed that only myofibers at the site of injection expressed beta-galactosidase enzyme. The persistence and strong expression of injected plasmid constructs suggest that zebrafish may be a simple and readily accessible system for direct muscle injection studies.

We report the first polymerase chain reaction (PCR) amplification of the complete mitochondrial genomes of a nemertean and a sipunculan worm in one piece using a recently published two-polymerase protocol for long and accurate DNA amplification. Successful amplification was achieved from nanogram quantities of both purified mitochondrial DNA (nemertean) and crude total DNA (sipunculan). This technique allows the rapid generation of sufficient quantities of entire mitochondrial DNAs for cloning and restriction fragment length polymorphism (RFLP) analyses, and thus will facilitate comparative studies of metazoan mitochondrial genomes.

Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications.

The phylogenetic position of the ancient family Pleurotomariidae within the Molluscan class Gastropoda, as well as the relationships of its Recent genera and species, were assessed using an iterative, two-gene (18S rDNA and cytochrome c oxidase I) approach to phylogeny reconstruction. In order to orient the Pleurotomariidae within Gastropoda, partial 18S rDNA sequences were determined for 7 pleurotomariid and 22 other gastropods that span the major groups within the class as well as for one cephalopod and two polyplacophorans, which serve as outgroups. Cladistic analyses of a sequence of approximately 450 base pairs (bp) near the 5' end of the 18S rDNA support the monophyly of the following higher gastropod taxa: Patellogastropoda, Vetigastropoda, Neritopsina, Apogastropoda, and its subclades Caenogastropoda and Heterobranchia. The 18S rDNA sequences and 579 bp of cytochrome c oxidase I (COI) analyzed separately and together, indicate that Pleurotomariidae are included within Vetigastropoda but comprise a clade that is the sister group to the other families referred to this order. Monophyly of the Pleurotomariidae is also supported by the unique presence of seven separate inserts (ranging in length from 1 to 68 bp) within the V2 variable region of the 18S RNA. Relationships of the genera and species within Pleurotomariidae are fully resolved using "total molecular evidence" consisting of partial sequences of 18S rDNA and COI and including data on length variation within the inserts.

The crustacean hyperglycemic hormones (CHHs) are released from the X-organ sinus gland complex and regulate the glucose level in the hemolymph. In the present study, we have isolated a complementary DNA that encodes a CHH (Pej-SGP-III) of the Kuruma prawn Penaeus japonicus to study its expression and the structure of its precursor. In a cDNA clone of 774 base pairs (bp), an open reading frame of 345 bp was found. The conceptually translated protein consists of a signal peptide (24 residues), a CHH-precursor-related peptide (CPRP, 17 residues), and Pej-SGP-III (72 residues). This CPRP in P.japonicus was homologous to, but significantly shorter than, those in other crustacean species. In a Northern blot analysis, the cDNA specifically hybridized to a seemingly single mRNA species (approximately 0.8 kb) in the eyestalk. This mRNA was not detected in the hepatopancreas or abdominal muscle.

Sequences of the internal transcribed spacer (ITS-1) ribosomal DNA region were compared among 88 soft-shell clams (Mya arenaria) from 12 sites (within three general areas) along the New England coast to determine whether populations were genetically heterogeneous. Two sequence variants were observed, with type 1 having a 3-nucleotide insertion and one point mutation relative to type 2. Allele-specific polymerase chain reaction (PCR); using primers specific to each sequence type, was performed to determine the distribution of individuals who had both allelic forms. DNA from soft-shell clams collected from three areas (Cobscook Bay, Maine; Gulf of Maine; and southern New England) were compared chi 2 analyses of allele-specific PCR results revealed no significant heterogeneity among the three population distributions.

An enriched zebrafish (Danio rerio) complementary DNA library was constructed for screening of ras-related genes, and a positive clone was isolated from one plate of 3 x 10(4) plaques. This clone, Zras-B1, carried an insert of 2592 base pairs (bp) with an open reading frame encoding a ras p21 protein of 188 amino acids. The deduced amino-terminal 86 amino acid residues and the carboxy-terminal CAAX binding motif are identical to mammalian ras. The full-length Zras-B1-encoded protein is most closely related to human N-ras (91% identity), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary screening data also indicate other ras genes in zebrafish, at least one of which is also transcribed in adults. A Zras-B1-related 3.1-kb transcript was found to be abundant in embryos from zygote through gastrulation, and may be maternally derived.

New strategies for embryonic manipulation have been developed in recent years through plant and animal research. However, research on marine invertebrate embryos has suffered from a lack of basic tools, such as microinjection. Here we present a technique developed for microinjecting eggs and embryos of the oyster Crassostrea gigas and the mussel Mytilus edulis. In experimental trials, approximately 40% of microinjected embryos survived. This technique was used to microinject beta-galactosidase, for which specific detection techniques were developed. A reporter construct (CMV-beta) based on a promoter of cytomegalovirus linked to the beta-galactosidase-encoding gene was then microinjected, and the expression level of this construct was monitored. The suitability of this technique is discussed in terms of its application to the manipulation of bivalve mollusks in pathology and genetics.

Two yeast strains, Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hfl CBS 8339, with a high capacity to colonize fish intestine were used in this study. The ability to adhere to crude mucus obtained from fish intestine was demonstrated for both strains. Scatchard analysis of the binding indicated a positive cooperativity for D. hansenii Hfl and absence of cooperativity for S. cerevisiae CBS 7764. In neither of the strains was adhesion extensively affected by reducing the hydrophobic interaction with p-nitrophenol, or by enhancing the hydrophobic interaction with ammonium sulfate. The adhesion was heat sensitive but resistant to trypsin treatment. We conclude that adhesion is mediated partly by specific mechanisms and partly by cell surface hydrophobicity.