Molecular marine biology and biotechnology最新文献

Three variable microsatellite loci have been isolated from the American lobster, Homarus americanus. In a population sample from the Gulf of Maine, the effective numbers of alleles (Ne) for the two most variable loci were 16.33 and 13.19, respectively. Reduced variability at all three loci was seen in the European lobster, H. gammarus, for which the maximum Ne was 4.00. The reduction in variability in H. gammarus is consistent with a bottleneck event. Inheritance analysis using H. americanus demonstrated segregation of codominant alleles and the absence of linkage. Null alleles were observed at two loci in inheritance studies. This study demonstrates that microsatellite loci should be useful in studying the population structure of clawed lobsters.

A highly repetitive interspersed DNA sequence (MRE) was isolated from the genome of medaka, Oryzias latipes, and characterized. Three distinct sequences of MRE were cloned and compared. The conserved sequences of MRE were approximately 220 bp in length. On average, one copy of MRE was present in every 153 kb, and the number of copies of MRE in the genome was calculated to be approximately 9800. MRE constituted approximately 0.14% of the genome. MRE-related sequences were not detected in carp (Cyprinus carpio) red-spot masu trout (Oncorhynchus masou macrostomus), masu salmon (Oncorhynchus masou masou), rainbow trout (Oncorhynchus mykiss), eel (Anguilla japonica), Arctic lamprey (Lampetera japonica), or yellowtail (Seriola quinqueradiata). MRE was randomly distributed in the medaka genome, indicating a high incidence of polymorphism in the five medaka inbred lines.

Atlantic haddock of Georges Bank are characterized by large fluctuations in population size and a recent collapse of the commercial fishery. DNA extracted from dried scales of Georges Bank haddock, archived by the U.S. National Marine Fisheries Service (NMFS), reveals significant heterogeneity in frequencies of four mitochondrial DNA control region haplotypes between 1975 and 1985 cohorts. Several processes may be responsible for this temporal variation, the most attractive hypothesis being that haddock from other geographic regions episodically contribute to the Georges Bank gene pool. Thus, the population of haddock spawning on Georges Bank may not be genetically discrete and, with respect to Atlantic haddock, Georges Bank may not be viewed as a closed system.

Transferrin is a monomeric iron-binding glycoprotein. To investigate the structure of the transferrin gene in teleosts, we prepared a genomic DNA library from the medaka (Oryzias latipes). The medaka transferrin gene was isolated, and its nucleotide sequence was determined. The full length of this gene is approximately 8.5 kb, and it is organized into 17 exons separated by 16 introns. The exons are similar in size to those in the genes for human transferrin, chicken ovotransferrin, and mouse and bovine lactoferrin. However, the introns are smaller than those previously reported in the transferrin family of genes. The 5'-flanking region of the medaka transferrin gene contains an estrogen-responsive element, glucocorticoid-responsive elements, an iron-responsive element, metal-responsive elements, and Sp1 binding sites. The transcription start point is located 35 bp upstream of the start codon.

Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas. Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes. Two culture media were used to establish primary cell cultures and tested as transfection media. Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions. In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species. Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters.

We developed three assays that detect genetic variation at single-copy nuclear loci in striped bass (Morone saxatilis). Because these assays are based on restriction enzyme digests of DNA amplified by the polymerase chain reaction (PCR-RFLP), they are easy to perform on large numbers of samples. Breeding trials demonstrated that the alleles identified in each of the three assays are inherited in a Mendelian fashion as codominant alleles at single-copy loci. To demonstrate the utility of these PCR-RFLP assays, we compared the genetic composition of striped bass populations from the Congaree River in South Carolina and from the Choptank River in Maryland. Allele frequencies were significantly different at the SB14 locus, suggesting that the two populations may be genetically distinct. Furthermore, during the development of the PCR-RFLP assays, we demonstrated that the GT(n) microsatellite-associated DNA regions (MSA regions) contained RFLPs at a frequency 9-fold higher than that observed for randomly chosen segments of DNA. If MSA regions proved to be variable in other organisms as well, they could provide a valuable source of intraspecific variation.

Plasmid vectors encoding the infectious hematopoietic necrosis virus (IHNV) nucleoprotein or glycoprotein gene under the control of a cytomegalovirus promoter were used to immunize rainbow trout (Oncorhynchus mykiss) against IHNV. The plasmid DNA was injected into the skeletal muscle of rainbow trout fry, and immunization was determined by the detection of virus-neutralizing and enzyme-linked immunosorbent assay antibody activity, and by protection against live virus challenge. Fish injected with the glycoprotein-encoding plasmid pCMV4-G, either alone or in combination with the nucleoprotein-encoding plasmid pCMV4-N, generated glycoprotein-specific and virus-neutralizing antibody responses. The vaccinated fish were also protected from subsequent IHNV challenge. Fish receiving pCMV4-N alone did not produce measurable virus-specific antibody and were killed by IHNV infection. These studies show that DNA vaccination will protect rainbow trout against the lethal effects of IHNV infection.

The amplification of DNA by polymerase chain reaction followed by restriction enzyme digestion was used to examine five anonymous single-copy nuclear DNA polymorphisms in 11 pair crosses for juvenile oysters (Crassostrea virginica). There was an overall 7% frequency of aberrant (non-Mendelian) offspring genotypes among 174 total pair-cross progeny. Strict Mendelian inheritance of alleles was observed for two of the five loci. Possible explanations for the aberrant offspring genotypes include nonamplifiable alleles owing to priming site polymorphism or aneuploidy, or paralogous amplification. Analysis of more pair crosses is needed to test these explanations.

An insulin-like growth factor (IGF) complementary DNA (cDNA) was isolated from a liver cDNA library of adult common carp, Cyprinus carpio. The identity of this cDNA clone was confirmed by nucleotide sequence determination. Sequence comparison with other vertebrate and rainbow trout IGFs showed that this cDNA encodes a particular subtype of IGF-I, IGF-IEa2. A reverse transcription-polymerase chain reaction (RT-PCR) assay showed that this IGF-I is expressed mainly in the liver (hepatopancreas) of adult common carp. Analyses of 26 other cDNA clones isolated from the same library (using a probe carrying the conserved region of the common carp IGF-IEa2 cDNA clone) showed that these clones represent different insert sizes of the same IGF-IEa2. In conclusion, IGF-IEa2 is the predominantly expressed IGF in the liver of adult common carp.

Expression of the firefly luciferase gene under the control of viral or fish promoters was observed in fish tissue after direct DNA injection of plasmid DNA. Plasmid DNA containing the firefly luciferase gene was injected into the skeletal muscle of rainbow trout (Oncorhynchus mykiss), and levels of luciferase activity were found to be dependent on the controlling promoter and the amount of injected DNA. Plasmids using the cytomegalovirus immediate early promoter (CMV-IEP) consistently produced the highest levels of luciferase activity. Maximal activity was observed five to seven days postinjection with 50 micrograms of DNA. This activity persisted in the tissues for as long as 115 days postinjection. When the DNA was examined up to two months postinjection, the predominant form was unreplicated, unintegrated DNA in linear and relaxed circular conformation. Expression of injected DNA was found predominantly within muscle cells along the injection path and in scattered muscle cells anterior to the injection site.