Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.
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.
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.
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 less than or equal to 10 cells per milligram of tissue by ethidium bromide detection, and this sensitivity was increased 10-fold by Southern blotting. There was a strong association (p <.001) between blood and ovarian fluid for the presence of bacteria in spawning salmon, and in 17.9% of the infected fish, bacteria were detected in blood but not in ovarian fluid. The ability to analyze multiple tissues, the reproducibility, and sensitivity of 16S RT-PCR make it a useful tool for both research and husbandry applications.
The estuarine teleost Fundulus heteroclitus exhibits some of the most remarkable genetic and morphologic transitions of any species along the Atlantic coast of the United States. Abrupt changes in gene frequencies at a variety of allelic isozymes have been identified at or near the Hudson River, and these changes are mirrored in morphologic characters. Analysis of mitochondrial DNA variation of four Atlantic coast populations identified two major mtDNA haplotype assemblages with a transition zone somewhere along the coast of New Jersey, but the concordance of patterns of mtDNA distributions and those evident in other genetic and morphologic features is unclear owing to the limited number of populations examined. In this study we extend the analysis of mtDNA variation to include 740 individual F. heteroclitus from 29 populations along the Atlantic coast and within the Chesapeake and Delaware Bays. The intent was to define more clearly the transition zone from southern to northern populations in reference to those already identified in allelic isozymes and morphologic characters. In addition, this study examined the distribution of mtDNA variation within two major estuaries (Chesapeake Bay and Delaware Bay) to determine if glacial relect populations of the northern form might still inhabit the upper reaches of these systems. The results indicate an abrupt transition in mtDNA haplotype frequencies in northern New Jersey, which is somewhat south of the transition zone indicated by allelic isozymes and morphologic features. The northern mtDNA haplotypes were also detected in the upper reaches of Chesapeake and Delaware Bays, but the frequency distribution was more clinal than that identified along the coast. On the basis of these data, we suggest that a single ancestral contact zone previously existed at least as far south as the mouth of the Chesapeake Bay prior to the last glaciation and that this zone has been unstable during the past several hundred thousand years.
We compared 413 nucleotides of the first subunit of the mitochondrial cytochrome oxidase c gene among 52 Tanner crabs from four locations of coastal waters of southern Alaska. Crabs from the Cook Inlet area possessed the largest haplotype diversity (0.933) as well as the largest number of haplotypes (11 of 18 observed). Crabs of southeastern Alaska had the lowest haplotype diversity (0.294) and only three haplotypes. Populations of Tanner crabs in waters of southwestern Alaska are apparently a mixture of both local and upstream haplotypes. Our data suggest the possibility that larvae of Tanner crabs are transported from east to west along the southern coast of Alaska by westwardly flowing currents. Possibly, declining stocks of crabs in western management areas are the result of overharvesting in eastern, upstream populations. The population in southeastern Alaska is probably genetically isolated with one predominant haplotype.
Commercially important oysters are often subject to mass mortality in the field. Rationalizing that heat shock can induce tolerance to stresses other than heat, we are exploring the possibility that this treatment might improve subsequent survival in the field. In characterizing the heat shock response in the laboratory, we found that heat shock of 37 degrees C for 1 h (HS) enabled Pacific oysters (Crassostrea gigas) to survive an otherwise lethal heat treatment (43 degrees-44 degrees C, 1 h) (LHT). This induced thermotolerance was retained for at least 2 weeks, the longest period examined. Stress proteins of the 70-kDa family were studied in gills by conventional autoradiography and immunoblotting techniques. Two constitutive isoforms of 77 and 72 kDa were up-regulated, and a 69-kDa protein was induced by HS, its synthesis occurring during and after that treatment. Synthesis of these three proteins was reduced to control levels within 2 days after HS, but their amounts remained higher than in control gills for 2 weeks following HS. Immunolocalization of proteins of the heat stock protein-70 (hsp-70) family in histologic sections of gills was consistent with results from Western blotting.
Isolation and characterization of microsatellite sequences from the genome of the black tiger prawn Penaeus monodon are described. Ninety-seven (GT)(n) and 16 (CT)(n) microsatellites were isolated from partial genomic libraries composed of 18,000 and 5250 clones, respectively. The genomic library screening indicated that (GT)(n) microsatellites are more abundant than (CT)(n) in P. monodon genome. The microsatellite sequences were classified into three categories, perfect, imperfect, and compound. The predominant categories found in P. monodon microsatellites are imperfect repeats for both (GT)(n) and (CT)(n). Very long repeat arrays were found in P. monodon microsatellite clones, which resulted in difficulties in primer design. Two microsatellite loci, CUPmo 18 and CUPmo 386, were successfully amplified. The number of alleles of each locus was preliminarily determined. For the CUPmo18 locus, Mendelian inheritance was tested by analysis of genotypic ratios in F-1 offspring and their parents. The results of this study demonstrate the presence of highly polymorphic microsatellite markers in P. monodon. These markers will be useful in population studies and parental determination in P. monodon. However, the low abundance and difficulties in obtaining a large number of usable microsatellite loci indicated that these markers may not be appropriate for use in genome mapping of this species.
Degenerate primers were designed from the amino acid sequence of the neuropeptide Pej-SGP-IV of the shrimp Penaeus japonicus. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using eyestalk complementary DNA of the sand shrimp Metapenaeus ensis. A partial cDNA that codes for a protein homologous to the neuropeptide Pej-SG-IV was cloned. The partial cDNA was used as a probe to screen the eyestalk cDNA library. Several cDNA clones with nucleotide sequence identical to the partial cDNA were isolated. The largest cDNA is 957 bp with an open reading frame consisting of a coding sequence 315 bp in length. The deduced amino acid of the neuropeptide consists of 77 amino acids and is preceded by a signal peptide of 28 amino acids. Because the deduced amino acid sequence of the shrimp cDNA is highly homologous to the Pej-SGP-IV of P. japonicus (which is molt inhibiting) and to other crustaceans' molt-inhibiting hormones (MIHs), the shrimp neuropeptide is tentatively called MeMIH. Northern blot analysis and RT-PCR showed that MeMIH is expressed in the postmolt, intermolt, and premolt stages of the shrimp eyestalks and the brain. Moreover, RNA message can also be detected in the nervous tissues of newly developed larvae. MeMIH is, however, not found in the muscle, swimming leg, and hepatopancreas. Results from genomic Southern blot analysis and amplification of the shrimp genomic DNA by polymerase chain reaction (PCR) suggest that a single copy of the MIH gene is present in the genome. The structural organization of the gene for the shrimp putative MIH is similar to that of the crab Charybdis feriatus.
We identified phytochelatins (PCs) as the major intracellular cadmium (Cd) chelators in the marine macroalga Kappaphycus alvarezii. Phytochelatin isolated from this alga showed a less complex thiol profile than that of yeast and higher plants. In the general structure of (Glu-Cys)(n)-Gly for PC, the n value was less than 3 for this alga. Algal PC-Cd complexes existed in the high molecular weight and low molecular weight forms. The high molecular weight form is rich in acid-labile sulfide. The PC content in algal fronds was directly correlated with the ambient Cd concentration as well as exposure time. After the Cd-treated algal fronds were transferred to Cd-free seawater, the algal PC content showed no significant change, while the total Cd content decreased.
The histone H3 (sH3) promoter of Atlantic salmon (Salmo salar) was cloned via polymerase chain reaction using primers designed from the rainbow trout (Oncorhynchus mykiss) promoter sequence. A comparison of the nucleotide sequence with the equivalent sequences from rainbow trout and sockeye salmon (Oncorhynchus nerka) revealed a high degree of conservation. In vivo expression analysis of the sH3 promoter was carried out in both rainbow trout and zebrafish (Danio rerio) embryos. A direct comparison of the sH3 promoter with the viral RSV promoter in rainbow trout resulted in stronger expression of the sH3 promoter. Furthermore, lacZ expression directed by the sH3 promoter was ubiquitous in several different cell types in developing zebrafish embryos. These results suggest that the sH3 promoter will be useful in transgenic studies in Atlantic salmon.
Cnidarians, one of the earliest lineages of metazoans, have clearly identifiable organs and a simple nervous system. The simplicity of this system affords fords an opportunity to study electrical and chemical transmission in its earliest stage of development. In this study the sea anemone Actinia spp. was examined for the presence of heterotrimeric G proteins. The bacterial toxin Bordetella pertussis was able to adenosine diphosphate (ADP) ribosylate a membrane-associated protein with a molecular mass of approximately 39 kDa, suggesting the presence of G(alpha)-subunits. In support of this finding, mastoparan, a direct activator of G proteins, induced a rapid tentacle contraction in a dose-dependent manner. Furthermore, antisera against conserved regions of alpha and beta-subunits of mammalian G proteins detected several candidate heterotrimeric G protein subunits. These three lines of evidence support the hypothesis that proteins involved in G-protein-coupled signal transduction pathways are expressed and functional in sea anemones.
To develop an antibody-based screen for epitopes involved in botryllid historecognition, BALB/c mice were immunized with whole Botryllus schlosseri colonies. Resulting monoclonal antibodies were screened for alpha or beta fusibility types using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. One monoclonal antibody (109) that recognized a polymorphic epitope was further analyzed by Western blotting. It binds a species-specific epitope localized to the atrial siphon and blood vessels. The epitope does not cosegregate with fusibility type. A complementary DNA clone encoding this antigen contains an endoplasmic reticulum retention motif. Polymorphism observed on Western blots was confirmed by Northern blot analysis. This antigen provides a new polymorphic marker that may be useful in studies of tunic formation.
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.
Successful preservation of tissue samples is a prerequisite for long field studies in remote areas. However, there is little published information concerning field preservation of marine invertebrate tissues for DNA analyses. This omission is significant because marine biodiversity is centered in the Indo-Pacific, where immediate DNA analysis is often impossible. Consequently, we used an assay based on polymerase chain reaction (PCR) to examine the effect of five storage solutions and three temperature regimens on the degradation of DNA from four common classes of marine invertebrates (Anthozoa, Gastropoda, Polychaeta, and Scyphozoa). Control samples were cryopreserved. Storage solution and the type of tissue preserved were the best predictors of preservation success. The length of time in storage and the storage temperature also affected the preservation of DNA. A field test demonstrates that a solution of dimethylsulfoxide and sodium chloride (DMSO-NaCl) preserves a wide range of tissues for DNA analyses and is very simple to use in remote field locations.
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.
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 complementary DNA (PjL26) containing an open reading frame for a protein of 144 amino acids was isolated from the decapod crustacean Penaeus japonicus. The conceptual PjL26 protein exhibits significant sequence similarities to ribosomal protein L26 in vertebrates. The level of the PjL26 messenger RNA in the tail fan did not change significantly during the molt cycle. This cDNA, therefore, may serve as a useful control probe in Northern analyses of stage-specific transcripts in P. japonicus and related crustacean species.
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.
