Molecular marine biology and biotechnology最新文献

Nine different transcription factors of the Strongylocentrotus purpuratus embryo, belonging to diverse structural families, were examined by high-resolution, two-dimensional gel electrophoresis. These factors had all been cloned previously, and antibodies against the recombinant proteins were available. The factors were visualized immunologically in nuclear extracts from 24-hour blastula-stage embryos. Remarkably, every one of the nine factors displayed multiple charge variants. Three factors, SpZ12-1, SpP3A2, and SpRunt-1, were studied at different stages of embryonic development. The prevalence and distribution of the variant isoforms of all three factors differed at each stage examined; and in all cases the complexity of the variants was greatest in the 24-hour blastula-stage extracts. The most complex set of variants was observed for SpP3A2, and phosphatase treatment demonstrated that some but not all, of the covalent modifications defining these variants are phosphorylations. As the transcription factors were chosen for this study merely on the basis of the availability of antibodies, we conclude that deployment of transcription factors in sea urchin embryos generally involves their covalent modification.

A fine-scale phylogenetic comparison was made among the symbionts of different genera of hydrothermal vent tube worms. These included Riftia pachyptila and Tevnia jerichonona, which inhabit sites along the east Pacific Rise, and Ridgeia piscesae from the Juan de Fuca Ridge. An analysis of restriction fragment length polymorphism (RFLP) was employed using three symbiont-specific gene probes: eubacterial 16S rRNA, RuBPC/O Form II, and ATP sulfurylase (recently cloned from the Riftia symbiont). Results indicated that all of the symbionts from the three different hosts were conspecific and the Riftia and Tevnia symbionts were indistinguishable over and 1800-km range. Significantly, this indicates that the symbionts have not co-evolved with their respective hosts, which are known to belong to separate families. This study strongly supports the conclusion that the symbionts are acquired de novo by each generation of juvenile tube worms from a common source in the surrounding sea water.

Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.

Five mircosatellite loci are characterized for the colonial ascidian Botryllus schlosseri. Within one population from Monterey, California, these loci have 3 to 17 alleles, observed heterozygosities from 0.40 to 0.63, expected heterozygosities from 0.52 to 0.84, and an overall paternity exclusion rate (QT) of 0.78. Three of the five loci demonstrated Mendelian patterns of inheritance in laboratory crosses. The size distribution of alleles suggests that most allelic diversity within these loci is generated by single-step and less frequently multistep mutations. However, several alleles may also have been generated by single based insertions or deletions. Mutation rates for the five microsatellite loci are less than 1 x 10(-2) per generation. Because or their highly polymorphic nature, these loci should prove useful for exploring issues of identity, kinship, population structure, and phylogenetics.

Nine different embryonic transcription factors interact at specific target sites in the cis-regulatory domain of the Strongylocentrotus purpuratus CyIIIa gene. We tested eight of these site sequences and show here that for every one a DNA-binding protein was present in unfertilized egg cytoplasm. The concentrations of active DNA-binding proteins per egg were estimated. We also present a new and convenient method for preparation of a material cytoplasmic fraction that retains these factors in active form.

In zebrafish it is possible to create viable diploid fish whose genomic DNA is derived only from the female parent (parthenogenesis) or, as was more recently shown, only from the male (androgenesis). Androgenesis requires holding zebrafish eggs in an inactivated state in vitro for an hour or more. Previously this was achieved by placing the zebrafish eggs in ovary fluid obtained from rainbow trout (Onchorhynchus mykiss) or coho salmon (Onchorhynchus kisutch). Here we report that adding bovine serum albumin (BSA) to Hank's buffered saline prevents zebrafish egg activation in vitro. Of the zebrafish eggs placed in Hank's saline plus 0.5% BSA, 85% +/- 8.7% were fertilizable after incubation for one hour at room temperature (23 degrees C). Longer incubations are possible but with lower efficiency of fertilization. This technique not only could facilitate androgenesis, but also might be useful when making transgenics by microinjection, when performing antibody or RNA injections before fertilization, or for studying the mechanisms of egg activation in zebrafish.

Three methods were used in succession to screen a whole adult zebrafish cDNA library for expressed p53-like genes. The sequences of the resultant clones describe an open reading frame 1122 nucleotides in length, with another 43 and 940 bases of 5' and 3' untranslated sequence, respectively. The deduced amino acid sequence of the zebrafish p53 protein is 63% identical to that of trout and 48% identical to that of human p53. Two of the three zebrafish clones overlap to span the entire reported cDNA sequence and are identical in their deduced amino acid sequence over their coincident length. The third clone contains a conservative amino acid change, as well as an inserted amino acid subsequently found to be at the junction of exons 2 and 3, suggestive of alternative splicing in the p53 mRNA for this species. Northern analysis demonstrated a zebrafish p53-related transcript to be present and most abundant in zygotes and early-cleavage embryos less than 1 hour after fertilization, thereafter declining to barely detectable levels at 48 hours. A similar temporal expression was detected for the zebrafish L-myc, known to be present in maternally derived RNA, whereas zebrafish N-myc and the zebrafish homologue of the murine T gene were not detectable prior to the onset of zygotic transcription.

The appearance of insulin-like growth factor I (IGFI) and II (IGF-II) mRNA was studied during rainbow trout embryonic development. Reverse transcription-polymerase chain reaction (RT-PCR), followed by Southern blotting and high-stringency hybridization with rainbow trout IGF-I and IGF-II cDNA probes, was used to detect all four forms of IGF-I mRNA and one form of IGF-II mRNA from whole-embryo total RNA isolated from a staged series. IGF-I and IGF-II mRNA were detected in unfertilized eggs. IGF-I and IGF-II mRNA were also detected during cleavage, gastrulation, and organogenesis, at hatching, during yolk absorption, and at feeding. IGF-IEa-1 and Ea-3 mRNA were detected in unfertilized eggs, while IGF-I Ea-4 was first detected at stage 9 when the zygotic genome is believed to become activated in rainbow trout. IGF-IEa-2 was not detected at any stage of embryonic development. IGF mRNA of immunoreactive peptides have been detected durin embryonic development in all vertebrates studied to date. Our results confirm the presence of IGF-I mRNA in teleost embryos. In addition, IGF-II mRNA is also present in teleost embryos. Our results suggest that the role IGF-I and IGF-II play during embryonic development may be conserved in vertebrates.

A novel intron-length polymorphism at the actin gene locus mac-1 is here reported and used as a genetic marker for population studies in mussels of the genus Mytilus. Two closely related genes subsequently identified as alleles, mac-1a1 and mac-1b1, from a genomic library of M. galloprovincialis were partially cloned and sequenced. They mainly differed from each other by a 65-bp insertion within their first intron. Polymerase chain reaction (PCR) primers were designed outside the insertion. The PCR analysis of 166 individual mussels from M. galloprovincialis and M. edulis populations revealed three size-classes of alleles or allelomorphs, two of which were of the expected sizes for mac1a1 and mac-1b1. One allelomorph was absent from M. edulis samples, although it was present at substantial frequencies in M. galloprovincialis populations. The frequencies of the two other allelomorphs significantly differed between M. galloprovincialis and M. edulis populations. The comparison of six mac-1 intron sequences over 277 bp showed at once that allelomorphs encompassed alleles differing from one another by substantial numbers of mutations, and that identical alleles were present in both M. galloprovincialis and M. edulis individuals, a probable result of the recent introgression between the two species.

Expression of a clam p53 homologue was examined in tissues of the soft-shell clam, Mya arenaria, from Beal's Island, Maine. Southern analysis reveals that p53, in this population, is a single copy gene. A 1.7 to 1.9-kb p53 mRNA was detected at very low levels in normal adult gonadal tissue. This transcript is similar in size to that of vertebrate p53 genes. RNAs were harvested from several tissues, including individual clam gonads during gametogenesis. These were hybridized in ribonuclease (RNase) protection assays to a p53 antisense probe designed from the clam p53 cDNA sequence. RNase protection profiles indicate that p53 mRNA is expressed in adductor muscle, gill, and gonads of both sexes. Although p53 mRNA is expressed throughout gametogenesis in mature male and female gonads, ovaries have significantly higher levels of expression. The significance of our findings to the study of normal clam gametogenesis and to etiology of gonadal tumors is discussed.