Molecular marine biology and biotechnology最新文献

We have cloned and sequenced cDNAs coding for the R1 and R2 proteins of ribonucleotide reductase from zebrafish (Danio rerio). This ribonucleotide reductase shows high amino acid sequence identity to those of other vertebrates. The R1 cDNA has a coding sequence of 2382 bp, yielding a 794 amino acid protein, and the R2 cDNA has a coding sequence of 1158 bp, yielding a 386 amino acid protein. The zebrafish R1 shows 94% similarity and R2 shows 91% similarity to the human R1 and R2, respectively. The similarity extends to intron positions, of which the equivalent of mouse R2 intron 3 has been studied.

The amount of the luciferin (substrate) binding protein (LBP) in extracts of the bioluminescent marine dinoflagellate Gonyaulax polyedra increases by about 10-fold during a 6-hour period spanning the end of the day- and the early night-phases, and then decreases by the same amount approximately 12 hours later. Previous studies have indicated that synthesis of the protein is regulated translationally. The experiments described here were undertaken to gain insight into the mechanism of this control. Evidence was obtained for the existence of mRNA-binding proteins that could have a regulatory function. In addition, circadian-related differences were observed in the total amount of mRNA as well as in the subcellular distribution of lbp mRNA.

Marine invertebrate collections have historically been maintained in ethanol following fixation in formalin. These collections may represent rare or extinct species or populations, provide detailed time-series samples, or come from presently inaccessible or difficult-to-sample localities. We tested the viability of obtaining DNA sequence data from formalin-fixed, ethanol-preserved (FFEP) deep-sea crustaceans, and found that nucleotide sequences for mitochondrial 16S rRNA and COI genes can be recovered from FFEP collections of varying age, and that these sequences are unmodified compared with those derived from frozen specimens. These results were repeatable among multiple specimens and collections for several species. Our results indicate that in the absence of fresh or frozen tissues, archived FFEP specimens may prove a useful source of material for analysis of gene sequence data by polymerase chain reaction (PCR) and direct sequencing.

Interspecific and intraspecific variation in the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA) was examined in two species of ahermatypic corals (lacking symbiotic algae and non-reef-building) with different dispersal characteristics, Paracyathus stearnsii (gamete-spawner with pelagic larvae) and Balanophyllia elegans (brooder with benthic larvae) from California. An approximately 300-bp region of the ITS-1 was amplified by polymerase chain reaction (PCR) and sequenced from populations at Pt. Loma, La Jolla, Monterey Bay, and Santa Catalina Island and compared for each species and compared with the same region from the hermatypic coral Favia lizardensis. There was a wide range of sequence variation in the ITS-1 region between the species, and these differences readily distinguished the taxa. Intraspecific comparisons of P. stearnsii individuals showed very little sequence variation (two polymorphisms) in the ITS-1 region. In contrast to P. stearnsii, we found 14 variable nucleotide sties in the ITS-1 region for B. elegans. Although there were no nucleotide sites that diagnostically separated central and southern populations, these findings indicate that the ITS-1 region is variable in B. elegans and a promising source for nuclear molecular markers in ahermatypic corals.

Amino acid sequences of chitinases have been determined in insects, plants, yeast, and bacteria, but not in crustaceans. We searched for chitinase-encoding cDNA in the kuruma prawn Penaeus japonicus by polymerase chain reaction (PCR) amplification of hepatopancreas cDNA using degenerate oligonucleotide primers derived from the two conserved regions of known chitinases. Using a PCR product as a probe, a cDNA clone was isolated. This clone contains an open reading frame for a protein (named Pjchi-1) of 572 amino acids that exhibits sequence similarities to known chitinases, especially to a chitinase from the tobacco hornworm Manduca sexta. Transcription of the mRNA was detected in the hepatopancreas but not in epidermal tissues.

Homeobox-containing genes are a phylogenetically widespread family of transcription factors that can regulate cell fates during embryogenesis. Two distinct homeobox gene sequences are described for the atentaculate ctenophore Beroë, the first homeoboxes to be identified in this phylum. Beroë homeobox fragments were cloned in a survey of genomic DNA using polymerase chain reaction (PCR). Parsimony, neighbor-joining, and maximum likelihood methods were used to infer the orthology of the ctenophore sequences to specific homeoboxes from higher metazoans including Drosophila, Caenorhabditis elegans, and humans. Cteno-paired appears most closely related to paired-typed homeoboxes. This is the first evidence of a paired-type homeobox in one of the so-called diploblastic animals. Cteno-Hoxl appears most closely related to members of the Hox class, particularly Antennapedia.

We report the development of a semiautomated multilocus genotyping system for Pacific salmon using four-color fluorescent detection of microsatellites. An initial screening of microsatellites was conducted on five species of Pacific salmon (Oncorhynchus spp.) and Atlantic salmon (Salmo salar) using 35 primer pairs developed from six species of salmonid. The number of loci that amplified varied by species from 11 (chum salmon) to 22 (chinook salmon). We then tested co-amplification of microsatellites in chinook, coho, and sockeye salmon and developed six-locus multiplex systems. The species-specific multiplex systems were applied to two populations using a sequencer/gene scanner (Perkin-Elmer Applied Biosystems, Inc. [ABI] 373A). The genetic variability at each locus was calculated to evaluate the utility of this system for genetic studies. Significant differences in allele frequencies were observed between populations in 14 of 18 pair-wise comparisons. Average heterozygosity ranged from 0.47 in Togiak River coho salmon to 0.75 in Dungeness River chinook salmon. Observed heterozygosities ranged from 0 at One mu 1 in Togiak River coho to 0.96 at Ssa85 in Dungeness River chinook. The probability of match (PM) for each six-locus multiplex system was 4.0 x 10(-10), 7.2 x 10(-8), and 3.2 x 10(-7) for chinook, coho, and sockeye, respectively. The average polymorphic information content (PIC) was 0.77, 0.56, and 0.60 for chinook, coho, and sockeye, respectively. The microsatellite loci used here show promise for high-resolution genetic studies of Pacific salmon such as fine-scale population analysis kinship, and parentage studies.

We have determined the complete sequence of the translation elongation factor EF1 subunit alpha (EF1 alpha) mRNA of zebrafish, and the 3'-untranslated sequence of EF1 alpha mRNA of halibut. The 5'-untranslated leader sequence of the EF1 alpha mRNA starts with a polypyrimidine tract. This feature is shared with the mRNAs for ribosomal proteins, where it affects the utilization of mRNA by ribosomes. However, the secondary structures of these leader sequences may differ. 5'-Polypyrimidine tracts of vertebrate EF1 alpha mRNAs participate in the formation of stable stem-loop structures, whereas those of 15 randomly chosen mRNAs for ribosomal proteins do not. This difference may provide a basis for differential control of translation for the two classes of mRNA. The 3'-untranslated sequences of vertebrate EF1 alpha mRNA have diverged little during evolution. Analyses of sequence and putative secondary structures suggest that both sequence-specific interactions and secondary structures may have contributed to sequence conservation.

Botryllus schlosseri is a colonial marine invertebrate that belongs to the subphylum Urochordata. Previously we analyzed the activity of a serine protease in this species, and cloned a tunicate chymotrypsin-like molecule. In the present study we further analyzed the protease activity of this animal, and found biochemical evidence also for specific trypsin-like activity. Subsequently we utilized a degenerate polymerase chain reaction (PCR) primer to clone two B. schlosseri cDNAs coding for two different putative trypsinogens, each 243 amino acids long, that differ within the coding region in 42 amino acids and 99 nucleotides. Both clones feature the characteristics of animal anionic trypsinogens. Sequence analysis of the tunicate putative trypsinogens revealed the invertebrate characteristics of three disulfide bridges, and higher similarity to invertebrate than to vertebrate trypsinogens. We therefore propose that the typical characteristics of vertebrate trypsinogens evolved after the divergence of Urochordates and Cephalochordates.

We have determined the complete sequence of an Atlantic cod (Gadus morhua) mitochondrial genome. The 16,696-bp genome contains the same 37 mitochondrial structural genes found in all other vertebrates analyzed, in an organization similar to that of the placental mammals. The cod mitochondrial DNA (mtDNA) contains variable numbers of a 40-bp heteroplasmic tandem repeat motif located in the major noncoding region, and the sequence presented here includes four repeats. Comparison among the major noncoding region in mtDNAs from different codfishes, as well as different stock samples of Atlantic cod, reveals conserved sequence features. The central region of the noncoding region as well as a spacer region located between the tRNAThr and tRNAPro genes appear to be variable in sequence, and are good candidates for high-resolution markers in population studies. Atlantic cod is the first marine bony fish whose mitochondrial genome is completely sequenced.