Molecular marine biology and biotechnology最新文献

Transferrin complementary DNAs were cloned from the livers of seven species in three genera of salmonids (kokanee salmon Oncorhynchus nerka, amago salmon Oncorhynchus masou ishikawa, masu salmon Oncorhynchus masou masou, Japanese char Salvelinus pluvius, brook trout Salvelinus fontinalis, lake trout Salvelinus namaycush, and brown trout Salmo trutta) subsequent to polymerase chain reaction amplification with primers derived from conserved regions of transferrin cDNA sequences. The transferrin cDNAs of the seven species of salmonids had sizes of 2.2 to 2.4 kb and encoded an open reading frame consisting of 691 amino acids with a putative signal peptide of 18 amino acids. The alignment of salmonid transferrin cDNAs showed a duplicated structure and conserved anion-binding residues, iron-binding residues, and cysteine residues for disulfide bridges. The deduced amino acid sequences of the seven salmonid transferrin cDNAs share 85% to 99% homology. A phylogenetic tree of amino acid sequences of transferrin cDNAs from salmonids showed that the relationship among the three genera of salmonids (Oncorhynchus, Salvelinus, and Salmo) is well correlated with that derived from classic morphologic and genetic analyses.

Muscle and melanoma tissue of fish in the genus Xiphophorus were examined for their ability to take up and express foreign DNA. Supercoiled plasmid DNA containing a firefly luciferase reporter gene with expression driven by the cytomegalovirus enhancer and thymidylate kinase promoter was directly injected into the muscle or melanoma of individual Xiphophorus. Expression levels gradually increased to a maximum at 6 days after injection in both tissues, and this level was maintained for at least 10 days after injection. In both muscle and melanoma, there was a clear relationship between dose injected and reporter gene activity, with maximal expression at a dose of 20 microg of plasmid injected. At higher doses expression levels declined, suggesting the possibility that the uptake mechanism can be inhibited by high concentrations of DNA. Histochemical localization using a beta-galactosidase construct revealed high expression of the enzyme in isolated muscle fibers. The activity of a second coinjected reporter gene, sea pansy (Renilla reniformis) luciferase, was highly correlated with the activity of the firefly luciferase reporter gene in both tissues (R2 >.940), suggesting that the majority of variation between samples results from variation in overall DNA uptake between individuals. When firefly luciferase activity is expressed as a function of activity of the coinjected reporter, the variation between samples is greatly reduced. As a result, small differences in activity between constructs can be detected. This demonstrates the usefulness of the system for gene expression analysis in vivo.

A lectin from Thai marine carb (Scylla serrata) hemolymph has been isolated and purified by affinity column chromatography and preparative electrophoresis. The amino acid composition and 10 amino-terminal residues have been deduced, and its reactivities have been studied using a biotin labeling technique. A method for the determination of sialoglycoconjugates in human serum is described using this lectin. The principle is based on the reaction between the sialoglycoconjugates and biotinylated lectin. The bovine submaxillary mucin (BSM) is immobilized on polystyrene microplate. The unknown sample or sialoglycoconjugate (BSM equivalent) standards, together with excess biotinylated purified lectin (B-lectin), are then added. The B-lectin that binds to the immobilized BSM is then incubated with the peroxidase-conjugated monoclonal antibiotin antibody, and the color that develops after the addition of enzyme substrate is determined by light absorption using a microplate reader. The assay is not only convenient and reliable, but also capable of measuring sialoglycoconjugates in solution at the submicrogram level. It was used in determining the sialoglycoconjugates in human serum from normal subjects and samples positive for carcinoembryonic antigen.

Variation at four microsatellite loci, Omy77, Ots100, Ots3, and Ots103, was characterized for three populations of sockeye salmon (Oncorhynchus nerka) found within 50 km of each other in the Barkley Sound region of Southwest Vancouver Island, British Columbia. We used a simple polyacrylamide gel electrophoresis technique to analyze microsatellite alleles amplified from total genomic DNA extracts of liver and archived scales. Classes from several years were examined for each population, with little significant interannual variability detected. Each microsatellite locus was highly polymorphic with 10 alleles at Omy77, 12 alleles at Ots100, 16 alleles at Ots3, and 17 alleles at Ots103; average observed heterozygosities were also high at 75%, 76%, 69%, and 85% respectively. Fst values and pairwise tests indicated that the populations had significantly different allele frequencies, suggesting that the three populations are reproductively isolated. Our results suggested that microsatellite loci can be analyzed with a simple technique to reveal population structure of sockeye salmon on a small geographic scale.

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H. curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

We describe a simple method for producing androgenetic haploid embryos in zebrafish. Mature eggs are expressed, and the maternal genome is inactivated by irradiation with short-wave ultraviolet light prior to in vitro fertilization. We demonstrate the absence of maternal genome in the resultant embryos using assays based on morphology and polymerase chain reaction. This technique for the production of androgenetic haploids will be extremely useful for identifying and recovering mutations as a complement to, or in place of, generating gynogenetic haploids.

A sensitive, reproducible assay for detecting Renibacterium salmoninarum in a variety of tissues, including blood, has been developed. This assay, based on reverse transcription-polymerase chain reaction (RT-PCR) of 16S ribosomal RNA, exhibited sensitivity to

Nucleotide sequences that surrounded ATG initiation codons were examined in jawless and cartilaginous fish complementary DNA sequences. Both thymidine and cytidine residues were underrepresented at positions near the initiation codon, while an extremely high frequency of purine nucleotides was observed at position -3. Statistical analysis (chi2) indicated that the greatest compositional bias occurred at nucleotide positions -3 and +4, and suggested that a relatively short consensus sequence surrounded AUG initiation codons of primitive fish genes. ATG triplets within 5' leader sequences were flanked by nucleotides different from those that surrounded ATG initiation codons. Dinucleotide frequency analysis indicated a deficiency in TA and an excess in AA around initiation codons. DNA sequence analysis suggested that low CpG conversion occurred 5' to the translation start of primitive fish genes. The conservation of consensus sequences around initiation codons of primitive fish genes underscores the importance of nucleotide composition for initiation of translation.

The genetic population structure of the European eel Anguilla anguilla L. was investigated by sequencing the mitochondrial D-loop region of 55 eels caught at different European locations. In total, 51 haplotypes were identified. Pairwise genetic distances ranged from 0% to 6.33% with an average value +/- SD of 3.01% +/- 1.18%, indicating little DNA differentiation among the European eel population. None of the node bifurcations of the neighbor-joining phylogenetic tree was strongly supported, suggesting that all European eels derive from a common genetic pool. The same result was obtained by a statistical test that confirmed the absence of geographic subdivision in the genotypes of the European eel population. The reported genetic homogeneity of the European eel is discussed in relation to different hypothetical life history scenarios.

DNA transfer techniques allow genetic manipulation of commercial fish. However, marine species have received little attention because of their difficult zootechnical requirements. The seabream (Sparus aurata) has become one of the most important species in the aquaculture of Mediterranean countries, and the development of suitable DNA transfer procedures represents a main step in its genetic improvement. To assess the response of the seabream to exogenous DNA, naturally fertilized eggs were injected with the plasmids pCMV-CAT, pCMVTklacZ, and pEGFP-N1, in supercoiled and linearized forms. Embryo and larval survival, DNA fate, and reporter gene expression were analyzed during early development. The survival results indicate that microinjection is an effective transfer method in spite of the unfavorable conditions. Linearized plasmids were more efficiently polymerized than supercoiled ones; however, no significant differences were detected either in their persistence or in their expression levels. Reporter gene expression was initiated after mid-blastula transition. The duration of transient expression varied between the promoter-gene combinations, and no integration of transgenes into fish chromosomes was detected. Results suggest that the main factor affecting the persistence and expression of DNA seems to be related to developmental processes. Among the markers used, CAT proved to be the most sensitive, but GFP had obvious methodologic advantages over the spatial marker lacZ. The usefulness of GFP for diagnosis of transgenesis is enhanced by the transparency of embryos and larvae in S. aurata.