Although changes in gene regulation may play an important role in adaptive evolution, there have been few attempts to investigate the molecular mechanisms responsible for adaptively significant variation in gene expression. Here we describe the mechanism underlying an adaptive difference in the expression of the lactate dehydrogenase-B gene (Ldh-B) between northern and southern populations of the fish Fundulus heteroclitus. Ldh-B regulatory sequences from northern and southern individuals, coupled to a luciferase reporter gene, were introduced into the livers of live fish. Deletion studies indicated that sequence changes between 400 and 500 bp upstream of the transcription start site resulted in a 2-fold difference in reporter gene transcription. These sequence changes can account for the previously observed 2-fold difference in Ldh-B transcription between populations. Variation in transcription factors did not play an important role. Sequences within the functionally important region resemble a mammary tumor virus glucocorticoid responsive element (MTV-GRE) in southern alleles, whereas northern alleles differ from the consensus by 1 bp. To test the hypothesis that this element is involved in the variation between populations of F. heteroclitus, we exposed transiently transgenic fish containing Ldh-B regulatory sequence/reporter gene constructs to handling stress or injected cortisol. Both treatments increased reporter gene transcription driven by southern alleles but not northern alleles, as expected if an MTV-GRE sequence were involved. This finding suggests that sequence variation in a GRE is the cause of the adaptive differences in Ldh-B gene expression between populations and demonstrates that small changes in gene regulatory sequences can have important evolutionary consequences.

Transgenic channel catfish (Ictalurus punctatus) containing salmonid growth hormone genes can grow 33% faster than normal channel catfish under aquaculture conditions. However, before transgenic catfish are released and utilized by the private sector, their genetic impact on the natural environment must be examined. Predator avoidance is one of the major fitness traits determining potential environmental risk. To determine the predator avoidance ability and growth performance of transgenic catfish in a natural habitat, various densities of transgenic and nontransgenic channel catfish were communally stocked in 0.04-ha earthen ponds without supplemental feeding. Largemouth bass (Micropterus salmoides) and green sunfish (Lepomis cyanellus) were stocked as predators. Nontransgenic fry had better predator avoidance than transgenic channel catfish when data were pooled (p <.01). When data were not pooled, nontransgenic catfish had better predator avoidance in six trials and transgenic individuals had better predator avoidance in four trials. There was no difference in predator avoidance in three trials. Overall predator avoidance was also better for nontransgenic individuals (p <.01) when the fish were evaluated as 3.5-g fingerlings, more clearly than as fry, as transgenic individuals were more vulnerable in 3 of 4 trials at this life stage. There was no significant difference in growth performance between transgenic and nontransgenic channel catfish in ponds without supplemental feeding. These findings indicate that transgenic channel catfish could be used for commercial aquaculture without affecting the natural environment. Although transgenic channel catfish may be released to nature by accident, any ecological effect would be unlikely because the increased susceptibility of transgenic channel catfish to predators would most likely decrease or eliminate the transgenic genotype.

We have isolated and characterized the 5′-flanking and part of the coding region of an actin gene from the red abalone Haliotis rufescens. There is high sequence homology between the abalone actin coding region and actins from other species. The sequence of this abalone actin is more closely related to vertebrate cytoplasmic actins than to muscle actins. RNase protection assays located the position of the transcription start point 66 bp upstream of the initiation codon. Promoter prediction by neural network located a TATA box 30 bp upstream of the transcription start point. A search with the SIGNAL SCAN program identified several potential transcription factor binding sites in the abalone sequence. These sites include sequences highly conserved in other actin promoters, like several putative CAAT and E boxes and a modified CArG box. Transfection assays with a construct containing the 5′ flanking region of the abalone actin coupled to a luciferase reporter gene showed that the promoter is functional in mammalian and fish cell lines, as well as in abalone gonad tissue. Expression vectors constructed with the abalone actin promoter will be useful for gene transfer studies into abalone and other mollusks.

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 have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Muscle and melanoma tissue of fish in the genus Xiphophorus were examined for their ability to take up and express foreign DNA. Supercoiled plasmid DNA containing a firefly luciferase reporter gene with expression driven by the cytomegalovirus enhancer and thymidylate kinase promoter was directly injected into the muscle or melanoma of individual Xiphophorus. Expression levels gradually increased to a maximum at 6 days after injection in both tissues, and this level was maintained for at least 10 days after injection. In both muscle and melanoma, there was a clear relationship between dose injected and reporter gene activity, with maximal expression at a dose of 20 microg of plasmid injected. At higher doses expression levels declined, suggesting the possibility that the uptake mechanism can be inhibited by high concentrations of DNA. Histochemical localization using a beta-galactosidase construct revealed high expression of the enzyme in isolated muscle fibers. The activity of a second coinjected reporter gene, sea pansy (Renilla reniformis) luciferase, was highly correlated with the activity of the firefly luciferase reporter gene in both tissues (R2 >.940), suggesting that the majority of variation between samples results from variation in overall DNA uptake between individuals. When firefly luciferase activity is expressed as a function of activity of the coinjected reporter, the variation between samples is greatly reduced. As a result, small differences in activity between constructs can be detected. This demonstrates the usefulness of the system for gene expression analysis in vivo.

We have investigated the mechanisms underlying differences in the transcriptional regulation of lactate dehydrogenase-B (Ldh-B) between northern and southern populations of a teleost fish, Fundulus heteroclitus. A 1-kb region immediately 5' of the gene was sequenced from populations throughout the species range. There were two major allele classes in the sample, one containing alleles from Maine and another containing those from Florida. Populations from intermediate localities contained both allele classes. Some individuals from Georgia had sequences intermediate between the two classes, representing either ancestral alleles or recombinants. Tests of neutrality were applied to determine whether observed variation was consistent with neutral expectations. Significant deviations from neutral expectations were detected for the 5' flanking region, but not for other loci. The functional consequences of flanking sequence variation were assessed by transfection of reporter gene constructs into cultured cells and injection into living fish. Consistent with observed variation in Ldh-B transcription rate between populations, significant differences in reporter gene activity were driven by flanking regions from northern and southern populations both in cell culture and in vivo. This functional differentiation, coupled with departures from neutral expectations, suggests that selection may have acted on the regulation of Ldh-B in F. heteroclitus.

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.

Twenty-three nests of kelp greenling (Hexagrammos decagrammus) were examined from British Columbia and central California. The average nest had four clutches of eggs that were associated with rock or rock and a biological substrate, encompassed an area of 1.7 m2, and was guarded by a male averaging 31 cm in length. Frequency distributions of egg-size classes were examined for 13 gravid females. Females were batch spawners capable of producing at least three clutches of eggs per spawning season. Variation in mitochondrial DNA among 107 individuals from 83 clutches representing 20 nests sampled from British Columbia and central California was analyzed. Different maternal contributors were found among clutches in 27% of nests from British Columbia and 55% of nests from California. ARENTAL care in fishes requires an expen

ABSTRACTSolar ultraviolet radiation (UVA + UVB) impairs photosynthesis in marine algae. Canopy blades of the giant kelp Macrocystis pyrifera (L.) C. Agardh are exposed to high levels of solar UV in the field. To determine the effects of UV radiation on photosynthesis in the giant kelp and to identify sites of UV damage, O2 evolution, reaction center organization, light harvesting, and energy transfer efficiency were measured in canopy blades that had been exposed to elevated levels of UV in the laboratory. UV treatment reduced both the light‐saturated rate and the light‐limited rate of photosynthesis by 50% but produced no significant change in the rate of dark respiration. A significant impairment of photosystem II (PSII) reaction center function was observed, suggesting that PSII is a major site of damage in chromophytes. Reduced quantum efficiency of photosynthesis and loss of energy transfer from light‐harvesting pigments (fucoxanthin, chlorophyll a, and chlorophyll c) to PSII indicate that the major light‐harvesting complex of M. pyrifera, the fucoxanthin‐chlorophyll protein complex (FCPC), was another site of UV damage. These measures provide the first evidence of a direct effect of UV radiation on specific sites in the photosynthetic apparatus of chromophytes and indicate that in situ fluorescence excitation analysis may be a simple means to detect UV stress in algae.

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.

Variation in enzyme expression may be an important mechanism for physiological and evolutionary adaptation. The Ldh-B locus in the teleost fish Fundulus heteroclitus is one of a very few loci for which an evolutionary difference in transcription rate between populations has been demonstrated. To begin to understand the molecular modifications that are responsible for altering transcription, we have characterized the Ldh-B proximal promoter using a combination of sequence analysis, transient transfection, and in vivo footprinting. The Ldh-B gene has several transcription start sites and a TATA-less, Inr (initiator of transcription motif) containing promoter with multiple Sp1-like motifs. Transfection experiments reveal that Sp1 sites, TCC repeats, and Inrs are functional components of the proximal promoter. We find substantial sequence variation between populations within the proximal promoter (250 bp from the transcription start sites) and footprinting analysis indicates that some of this sequence variation is associated with differential protein binding to the apparent TFIID binding site and Sp1 sites. Together, these data suggest that variation in the Ldh-B proximal promoter may play a role in the observed difference in transcription rates between northern and southern populations of F. heteroclitus.

Gene duplication has produced two lactate dehydrogenase (LDH) isozymes, LDH-A and LDH-B, that are found in essentially all vertebrates. On the basis of the biochemical properties of the LDH-A and LDH-B isozymes, it has been suggested that each locus is orthologous among all vertebrates. However, phylogenetic studies have not supported a common evolutionary history among the LDH-A isozymes, particularly when those from lower vertebrates are examined. We present here the sequence of a muscle-type LDH from Fundulus heteroclitus, a teleost fish for which the LDH-B sequence has been determined and shown to be unrelated phylogenetically to tetrapod LDH-A isozymes. Although the sequence of the teleost muscle LDH shares certain features with the LDH-A of tetrapods, phylogenetic analyses do not support an orthologous relation among the LDH-A isozymes of teleost fish and tetrapod vertebrates.

Fundulus heteroclitus in the estuary of the Guadalquivir River in southern Spain competes with a native killifish, Aphanius iberus. Cytochrome b sequence analysis shows that the Spanish individuals are derived from the northern of two possible American populations of F. heteroclitus

We have isolated and characterized several recombinant lambda phage clones carrying growth hormone (GH) cDNA of striped bass (Morone saxatilis). Nucleotide sequence and the predicted amino acid sequence of sbGH was determined from a recombinant clone carrying the longest cDNA insert. The sbGH cDNA encodes a pre-hormone of 204 amino acid residues. Comparison of the predicted amino acid sequence of sbGH with those of other vertebrates revealed different degrees of sequence identity: approximately 98% with European sea bass; 90% with bluefin tuna; bonito and red seabream; 71% with winter flounder; 64% with salmonids; 55% with carp; and 38% with human. Expression of the mature sbGH cDNA (without the signal peptide sequence) in E. coli cells under regulation of the lambda phage PL promoter produced a polypeptide of 20 kDa. Following renaturation, this recombinant hormone was shown to be biologically active in a radioreceptor competition binding assay and in the induction of hepatic insulin-like growth factor I (IGF-I) mRNA synthesis in vivo.

Here we report a simple method of in vivo footprinting for the detection of DNA-protein interactions in the liver of a small teleost fish, Fundulus heteroclitus. This method allows the determination of these interactions in nuclei isolated from intact liver, obviating the need for cell culture. Cells in culture often do not respond to environmental cues in the same way as do tissues within the intact organism and therefore may be inappropriate for the study of certain adaptive responses. Furthermore, cell lines are available for only a small number of marine organisms. This technique may therefore be of general utility for the study of gene regulation in a wide variety of marine organisms.

Recombinant plasmid containing the Drosophila beta-actin promoter coupled to a beta-galactosidase cassette was linearized and introduced in fertilized eggs of the red abalone (Haliotis rufescens) by electroporation. Fertilized abalone eggs tolerated electroporation well with larval survival rates between 70% and 84% of that for non-electroporated siblings. Dot blot and Southern blot analysis were used to detect if abalone retained the foreign gene at various developmental stages. The inserted construct was retained in 70% to 100% of all abalone sampled with an average of 72% retention in the three- to seven-month-old juveniles. Maximal DNA uptake and retention was observed in abalone electroporated at 30-40 min after fertilization. Southern hybridization analysis suggested that the inserted vector was in head-to-tail concantermers integrated in the abalone genome. This preliminary study demonstrates that electroporation is an efficient means of transferring foreign DNA into abalone embryos.

Six members of a multigene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex from female gametophytes of the brown alga Macrocystis pyrifera have been cloned and characterized. The deduced amino acid sequences are very similar to those of fucoxanthin chlorophyll binding proteins (Fcp) from the diatom Phaeodactylum tricornutum and exhibit limited homology to chlorophyll a/b binding (Cab) polypeptides from higher plants. The primary translation products from the M. pyrifera fcp genes are synthesized as higher molecular weight precursors that are processed prior to their assembly into the Fcp complex. The presumed N-terminal 40-amino acid presequence of the Fcp precursor polypeptide has features resembling that of a signal sequence. This presequence may be required for the protein to transverse the endoplasmic reticulum that surrounds the plastid in brown algae. A subsequent targeting step would be required for the protein to cross the double membrane of the plastid envelope. M. pyrifera fcp transcripts are of two sizes, 1.2 and 1.6 kb. The size difference is accounted for by the length of the 3' untranslated region, which can be up to 1000 bases. Transcript abundance's of members of the fcp gene family are dependent on light quantity, light quality, or both. Transcript levels of one gene increased approximately five- to tenfold in thalli grown in low intensity relative to high intensity white or blue light. Transcripts from this gene also significantly increase in red light relative to blue light at equivalent light intensities.

F1 transgenic common carp, Cyprinus carpio, containing rainbow trout growth hormone gene, pRSVrtGH1 cDNA were compared to non-transgenic full-siblings for body composition. Percent protein was higher (P < 0.05), for transgenic individuals than for controls (19.5 vs. 18.1). Percent fat was lower (P < 0.05) for transgenic common carp (3.3), than for non-transgenic controls, (3.8). Transgenic individuals had lower (P < 0.05) moisture content (70.9%), than controls (75.8%). Of the 18 amino acids examined the transgenic genotype had higher (P < 0.05) levels for the amino acids aspartic acid, cystine, glutamic acid, histidine, lysine and threonine. Observed values for transgenic individuals were higher for all amino acids except glycine, alanine, proline and tryptophan. The actual ratio among the 18 amino acids remained the same except the percentages increased (P < 0.05) from 15.4 to 15.8%, and 2.7 to 3.0% for glutamic acid and histidine and decreased from 6.6 to 4.9% for glycine, respectively, for transgenic individuals relative to controls. The essential amino acid ratios (AE ratios) of lysine and histidine were higher (P < 0.05) in transgenic compared to control fish muscle. The C18:1 fatty acids dominated the fatty acid composition of both groups and fatty acid levels were unchanged in transgenic and control common carp.