Molecular marine biology and biotechnology最新文献

We describe the construction of amplification primers designed to target a portion of the mitochondrial cytochrome b locus in a variety of molluscan taxa. Combinations of two sets of primers successfully amplified cytochrome b from several species of gastropods, bivalves, and cephalopods. Sequence analysis of these amplified products revealed nucleotide diversity in small samples within several of these taxa. We discuss the utility of these primer sets for studies of intraspecific phylogeny in mollusks and potentially other invertebrates.

The gene coding for proliferating cell nuclear antigen (PCNA) was identified in Dunaliella tertiolecta (Chlorophyceae) and Isochrysis galbana (Prymnesiophyceae). Southern blot hybridization using a PstI fragment of rat PCNA gene (pCR-1) as a probe showed that there is apparently a single copy of this gene per haploid genome in both species. On the Northern blot pCR-1 probed a single messenger RNA for each species of a molecular size close to rat PCNA mRNA (1.1 kilobases [kb]). Polymerase chain reaction (PCR) with a set of degenerated primers produced a fragment of about 610 base pairs [bp] from genomic DNA of both species; the PCR products appeared close in size to the amplified from rat PCNA and hybridized to the pCR-1 probe. Further analysis with reverse transcription-PCR (RT-PCR), cloning, and sequencing revealed a complementary DNA of a similar size (616 and 576 bp) that possesses an open reading frame encoding 205 and 192 amino acids, respectively, for Dun (D. tertiolecta) and Iso (I. galbana). Surprisingly, the polypeptides deduced from the two cDNA shared no higher homology to each other (71%) than to animals such as Xenopus (Dun 72%; Iso 73%), rat (Dun 73%; Iso 74%), and human (Dun 73%; Iso 74%), and to higher plants such as soybean (Dun 78%; Iso 72%), Zea mays (Dun 77%; Iso 73%), and rice (Dunn 77%; Iso 72%), although D. tertiolecta has a higher homology (77%) to the Prasinophyceae alga Tetraselmis chiu than does I. galbana (71%). The homology to PCNA in budding and fision yeasts (63% and 53%, respectively) is also lower than to animals and higher plants. It is thus suggested that with regard to PCNA genes, the three algae are as different from each other as they are from higher plants and animals. In a partially synchronized exponential culture of D. tertiolecta grown with a photocycle of 12 h light and 12 h dark, the abundance of the transcript appeared to be low at hours 3 and 9 (hour 0 = the onset of light period), and increased about 2- to 3-fold at hours 15 and 21 (i.e., during the dark period). Western blotting and immunofluorescence analysis on concurrent diel samples showed an over 2-fold increase in PCNA protein abundance (in proportion to total cellular protein) and the percentage of cells labeled by PCNA antibody. A similar trend was found for I. galbana grown under the same conditions. The results suggest that the gene transcription was in pace with PCNA synthesis, which was lower in the light period when G1 phase was dominant and higher in the dark period when S (and probably G2 and early M) phase was dominant, and that the expression of this gene may be regulated at the transcriptional level in these two algae.

Crustaceans are a major cause of seafood allergy. Recent studies have identified tropomyosin as the major allergen in shrimp. However, such data are lacking in other crustaceans. In the present study lobster allergens were identified and characterized by molecular cloning, sequencing, and expression. An IgE-reactive complementary DNA clone of 2 kilobase pairs (kb) was identified by screening an expression library of the spiny lobster Panulirus stimpsoni using sera from subjects with crustacean allergy. Expression and sequencing of this clone showed that it has an opening reading frame of 274 amino acids, coding for a 34-kDa protein designated as Pan s I. In addition, we expressed the fast muscle tropomyosin from the American lobster Homarus americanus and found that this protein, coined Hom a I, was also recognized by IgE from patients with crustacean allergies. The deduced amino acid sequences of Pan s I and Hom a I, which are the first identified lobster allergens, show significant homology to shrimp tropomyosin. Sera from subjects with crustacean allergies, when preabsorbed with recombinant proteins Pan s I or Hom a I, lost their IgE reactivity to muscle extract of P. stimpsoni and H. americanus. Preincubation of crustacean allergy sera with the recombinant shrimp tropomyosin Met e I also removed their IgE reactivity to lobster muscle extracts. The results suggest that patients with allergic reactions to crustaceans have common and possibly cross-reactive IgE-reactive epitopes in lobster and shrimp.

Expressed sequence tags (ESTs) were obtained from Japanese flounder (Paralichthys olivaceus). We present the results of single-pass sequencing of 493 ESTs from 350 clones from liver cDNA and 57 ESTs from 41 clones from spleen cDNA. Sequences of the cDNA clones were compared with sequences in the GenBank database. Two hundred and two clones (51.7%) appeared to be completely unknown and are likely to represent newly described genes, whereas 189 clones (48.3%) were identified based on matches to sequences in the databases. Three of the unidentified sequences were isolated from both the liver and spleen cDNA libraries. However, there were no identical sequences between liver and spleen clones.

A cDNA clone of rainbow trout (Oncorhynchus mykiss) transferrin was obtained from a liver cDNA library. The 2537-bp cDNA sequence contained an open reading frame encoding 691 amino acids and the 5' and 3' noncoding regions. The amino acid sequences at the iron-binding sites and the two N-linked glycosylation sites, and the cysteine residues were consistent with known, conserved vertebrate transferrin cDNA sequences. Single N-linked glycosylation sites existed on the N- and C-lobe. The deduced amino acid sequence of the rainbow trout transferrin cDNA had 92.9% identities with transferrin of coho salmon (Oncorhynchus kisutch); 85%, Atlantic salmon (Salmo salar); 67.3%, medaka (Oryzias latipes); 61.3% Atlantic cod (Gadus morhua); and 59.7%, Japanese flounder (Paralichthys olivaceus). The long and accurate polymerase chain reaction (LA-PCR) was used to amplify approximately 6.5 kb of the transferrin gene from rainbow trout genomic DNA. Restriction fragment length polymorphisms (RFLPs) of the LA-PCR products revealed three digestion patterns in 22 samples.

The transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. However, the ectopic GH levels to optimize growth acceleration in fish, and specially in tilapia, are not known and must be determined experimentally. The tilapia GH (tiGH) cDNA was used to construct chimeric genes expressing different levels of tiGH in vitro and in vivo. These constructs were used to generate four lines of transgenic tilapia by microinjection into one-cell embryos. Different patterns and levels of ectopic expression of tiGH and IGF were detected in organs of transgenic tilapia by RNA or protein analysis. The two lines with lower ectopic tiGH mRNA levels were the only ones showing growth acceleration, suggesting that the expression of ectopic tiGH promoted growth only at low expression levels. The effect of higher ectopic tiGH levels resembled the physiologic situation of low condition factor and permitted us to postulate a model for growth acceleration in transgenic tilapia expressing ectopic tiGH.

Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.

Sponges are suspension-feeders that are devoid of body cavities. Phagocytosis is the major route of nutrition in these animals. In an attempt to understand protein digestion, cathepsin was identified in crude extracts from the sponge Geodia cydonium. This enzyme was purified from lysosomes by a two-step procedure--pH precipitation and FPLC separation--to apparent homogeneity; it showed an M(r) of 26,000. Inhibitor as well as substrate studies showed that the sponge cathepsin belongs to the subfamily L of these cysteine proteases. The complete cDNA coding for cathepsin L was isolated and characterized. The deduced aa sequence contains 322 residues, has an M(r) of 36,085, and shows the characteristic signatures known from other cathepsins of the L subfamily: e.g., cleavage site for the proregion, the ERFNIN motif, and the conserved regions forming the catalytic triad of cysteine proteases. Phylogenetic analyses revealed that the sponge sequence groups with the cathepsin L subfamily and branches off first from the other metazoan members. The sponge sequence shows high homology to that isolated from Dictyostelium discoideum and only low similarity to the protozoan cathepsins L from Paramecium tetraurelia and Tetrahymena thermophila. From the data presented it is concluded that cathepsin L is the major digestive protease in sponges.

A medaka liver cDNA library was constructed in lambda ZAPII. The number of clones in this library is approximately 5 x 10(6). Three hundred sixty-one clones were randomly selected and, of these, 33 clones with over 1 kb were sequenced. These sequences were compared with GenBank and the dbEST (Re.96.0). Twenty-five clones of these 33 clones encoded 18 different genes and 2 different expressed sequence tags (ESTs). Sequences of 10 of the 18 clones had not previously been reported in fish. The codon usage of medaka genes is similar to that of Xenopus, mouse, and human genes, but not similar to that of yeast and Escherichia coli genes.

Lysozyme is a widely distributed enzyme located in the serum, skin mucus, and other organs of fish, which is responsible for catalyzing the hydrolysis of the cell walls of most bacteria. A c-type of lysozyme cDNA was cloned from a kidney cDNA library of the Japanese flounder (Paralichthys olivaceus). The cDNAs consisted of 612 bp, which coded for 143 amino acid residues. The deduced amino acid sequence of Japanese flounder c-type lysozyme possessed 72.9%, 57.4%, and 65.4% identities with rainbow trout, chicken, and human c-type lysozymes, respectively. Comparison of the c-type lysozymes showed that the catalytic residues, the residues binding to sugar chains, and cysteine residues were completely conserved. Northern blot analysis indicated that the c-type lysozyme gene is apparently transcribed in the head kidney, posterior kidney, spleen, brain, and ovary of healthy flounder. When flounder were experimentally infected with Edwardsiella tarda, quantities of the c-type lysozyme mRNA increased in the head kidney, spleen, and ovary of the flounder.