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G L Fletcher

Publications and source records attributed to G L Fletcher.

At least 19 recordsLinked to original sources
PubMed1996

Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prosequences.

Distinct antifreeze polypeptides (AFP) were isolated from the skin of the winter flounder, Pleuronectes americanus, by gel filtration and reverse phase high performance liquid chromatography. In parallel, several cDNA clones were isolated from a skin cDNA library using a liver AFP cDNA probe. Both protein and DNA sequence analyses indicate that flounder skin contains several distinct but homologous alanine-rich AFPs. Although the skin type AFPs contain 11 similar amino acid repeats found in the secretory liver type AFPs, the skin type AFPs are mature polypeptides lacking both the signal and prosequences, indicating that they may function intracellularly. The skin type AFP is significantly less active in thermal hysteretic activity than the liver type AFP. Genomic Southern analysis indicates that like the liver type AFP genes, there are multiple copies (30-40 copies) of skin type AFP. Although the liver type AFP genes are specifically expressed in the liver and to a lesser extent in intestine, the skin type AFP genes are expressed in all tissues examined including the liver and abundantly in exterior tissues, i.e. skin, scales, fin, and gills, suggesting an important protecting role in these exterior tissues.

Alanine↗
PubMed1995

The antifreeze protein genes of the winter flounder, Pleuronectus americanus, are differentially regulated in liver and non-liver tissues.

The synthesis of winter flounder (Pleuronectus americanus) antifreeze protein (AFP) mRNAs in the liver is seasonally regulated by the pituitary gland. With the recent discovery that AFP mRNAs are also present in several non-liver tissues, the aim of the present investigation was to compare the regulatory mechanisms of AFP genes in liver and non-liver tissues. Northern blot analyses indicate that the level of liver AFP mRNA undergoes a several hundred fold difference between the winter and summer months, while AFP mRNAs from gills and kidneys exhibit only a modest 5-10 fold seasonal variation. As expected, the liver AFP mRNA in the hypophysectomized fish was increased by over 40 fold. However, no significant increase was observed for the non-liver AFP mRNAs upon hypophysectomy. These investigations suggest that AFP mRNAs in liver and non-liver tissues are differentially regulated.

Animals↗
PubMed1993

Herring antifreeze protein: primary structure and evidence for a C-type lectin evolutionary origin.

A complementary DNA (cDNA) for a type II antifreeze protein (AFP) was isolated from an Atlantic herring liver cDNA library and sequenced. The predicted protein sequence is homologous to those of the type II fish AFP from smelt and sea raven; it is also homologous to the carbohydrate recognition domains (CRD) of calcium-dependent (C-type) lectins and similar domains in lectin-like proteins. Herring belong to the infradivision Clupeomorpha, which is distinct from the Euteleostei to which all other AFP-producing fish belong. The occurrence of type II AFP in widely divergent fish groups and their homology to C-type lectin CRDs suggest that type II AFPs evolved from these lectins. Amino acid residues forming the hydrophobic cores of rat mannose-binding protein A (MBP-A) that are conserved in character among C-type lectins are also conserved in the herring AFP. The herring AFP also requires Ca2+ for thermal hysteresis activity. These results suggest that herring AFP is structurally and functionally similar to the CRDs of C-type lectins and related domains in other proteins.

Amino Acid Sequence↗
PubMed1992

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.

Amino Acid Sequence↗
PubMed1992

Growth enhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone gene construct.

We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

Animals↗
PubMed1992

Fish antifreeze proteins block Ca entry into rabbit parietal cells.

Many fish and insects have adapted to life at subfreezing temperatures by evolving so-called antifreeze proteins (AFP) that noncolligatively depress the freezing temperatures of aqueous solutions without affecting the melting temperature. AFP have been thought to function solely as antifreezes. Recently, however, we discovered that AFP also protect mammalian cells and organs from damage caused by exposure to hypothermic (above freezing) temperatures. It has been proposed that hypothermic damage is caused by changes in intracellular ionic content due to a reduction of active transport that is required to balance passive ion transport across cell membranes. Given this possibility, we tested whether AFP isolated from the Newfoundland ocean pout might reduce the Ca ion permeability of a mammalian cell, the rabbit gastric parietal cell, which has been particularly well studied in terms of Ca transport and signaling. Digital image processing of the Ca-sensitive fluorescent indicator fura-2 was used to measure intracellular free Ca in these cells. During stimulation with the cholinergic agonist carbachol, AFP inhibited passive Ca entry across the cell membrane without interfering with either the release of Ca from internal stores (indicating that the carbachol receptor and other signaling events were operational) or the normal active rates of Ca efflux from the cell (indicating that Ca pumping was also still intact). These results suggest that, in addition to their actual antifreeze properties, AFP may also help to confer cold tolerance in animals by preventing passive Ca entry into epithelial cells.

Action Potentials↗
PubMed1992

Inhibition of Ca2+ and K+ currents by "antifreeze" proteins.

For the last two decades, the research on fish "antifreeze" proteins has focused exclusively on their ability to depress noncolligatively blood plasma freezing points, presumably by binding to ice crystals. We report evidence that antifreeze polypeptides from the winter flounder (Pseudopleuronectes americanus) have another special property, the ability to block ion channels. In experiments with porcine granulosa cells we show, using the patch-clamp technique in the whole cell configuration, that these proteins suppress effectively calcium and potassium currents. The results of dose-response studies indicate a protein-protein interaction mechanism.

Animals↗
PubMed1991

Hypothermic protection--a fundamental property of "antifreeze" proteins.

For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4 degrees C for 24 h in the presence of type I alanine rich alpha helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that cold-sensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins.

Animals↗
PubMed1991

Vitellogenin gene transcription is not under strict estrogen control in winter flounder.

Although it is almost axiomatic that vitellogenin gene expression is under exclusive control of estrogen in oviparous animals, our results with winter flounder demonstrate that vitellogenin gene transcription in females can continue independent of estradiol. Winter flounder were hypophysectomized in January, i.e. several months after the onset of vitellogenesis. Thirty or more days after hypophysectomy, all fish had negligible levels of estradiol in the serum, and yet vitellogenin gene transcription was quite active in the liver. Our results also suggest that a pituitary factor may be involved in the normal repression of the vitellogenin gene.

Animals↗
PubMed1989

Regulation of antifreeze protein production in winter flounder: a unique function for growth hormone.

Salmon pituitary extract and the protein fraction unabsorbed on concanavalin A-Sepharose, the carbohydrate-poor fraction, depressed plasma levels of antifreeze proteins (AFP) when the pituitary fractions were administered to flounder in late fall or winter. The active pituitary protein occurred in the fraction with a mean molecular weight of 25,000. The two major isohormones of growth hormone (GH) were the only biologically active proteins identified from the pituitary. Hypophysectomized flounder synthesize AFP in the spring and the two isohormones of GH suppress the synthesis. The fraction of flounder pituitaries containing putative GH depressed flounder plasma levels of AFP in late fall.

Acclimatization↗
PubMed1989

Antifreeze protein gene transcription in winter flounder is not responsive to temperature.

Although the endogenous rhythm of antifreeze protein gene expression in winter flounder is primarily regulated through the pituitary, the effect of water temperature on the annual cycle is poorly understood. In order to determine the specific effects of temperature on antifreeze gene transcription we did a series of experiments with intact and hypophysectomized winter flounder kept at various temperature regimes. Our results demonstrate that temperature shifts do not induce or suppress antifreeze gene transcription as determined by "run-on" transcription assays or by Northern blot analysis of liver mRNA in hypophysectomized flounder. However, warm temperature reduces the amount of antifreeze protein in the plasma, and apparently reduces the half-life of antifreeze protein mRNA.

Animals↗
PubMed1988

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus.

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.

Amino Acid Sequence↗
PubMed1988

Differential amplification of antifreeze protein genes in the pleuronectinae.

The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10-12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.

Amino Acid Sequence↗
PubMed1988

Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Amino Acid Sequence↗