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Garth L Fletcher

Publications and source records attributed to Garth L Fletcher.

8 recordsLinked to original sources
PubMed2006

Characterization and multi-generational stability of the growth hormone transgene (EO-1alpha) responsible for enhanced growth rates in Atlantic Salmon.

Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an "all fish" gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5' promoter and 3' termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1alpha) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1alpha integrant was organized as follows: base pairs 1580-2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3' region, and the first 1678 bp of the ocean pout antifreeze 5' region. Sequence analysis of the EO-1alpha integrant and genomic flanking regions in F2 and F4 generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1alpha transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.

Animals↗
PubMed2005

Type I antifreeze proteins: possible origins from chorion and keratin genes in Atlantic snailfish.

Type I antifreeze proteins (AFPs) are alanine-rich alpha-helical polypeptides found in some species of right-eye flounders, sculpin, and snailfish. In this study, a shorthorn sculpin skin type I cDNA clone was used to probe an Atlantic snailfish liver cDNA library in order to locate expressed genes corresponding to snailfish plasma AFPs. Clones isolated from the cDNA library had sections with substantial amino acid and nucleotide sequence similarity to snailfish type I AFPs. However, further analysis revealed that the positives were actually three different liver-expressed proteins-two were eggshell proteins, while the third was a type II keratin. We propose that a shift in reading frame could produce alanine-rich candidate AFPs with possible antifreeze activity or ice crystal modification properties. Furthermore, it is plausible that one or more of the liver-expressed proteins represent the progenitors of snailfish type I AFPs.

Amino Acid Sequence↗
PubMed2005

Hyperactive antifreeze protein in flounder species. The sole freeze protectant in American plaice.

The recent discovery of a large hyperactive antifreeze protein in the blood plasma of winter flounder has helped explain why this fish does not freeze in icy seawater. The previously known, smaller and much less active type I antifreeze proteins cannot by themselves protect the flounder down to the freezing point of seawater. The relationship between the large and small antifreezes has yet to be established, but they do share alanine-richness (> 60%) and extensive alpha-helicity. Here we have examined two other righteye flounder species for the presence of the hyperactive antifreeze, which may have escaped prior detection because of its lability. Such a protein is indeed present in the yellowtail flounder judging by its size, amino acid composition and N-terminal sequence, along with the previously characterized type I antifreeze proteins. An ortholog is also present in American plaice based on the above criteria and its high specific antifreeze activity. This protein was purified and shown to be almost fully alpha-helical, highly asymmetrical, and susceptible to denaturation at room temperature. It is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins, the latter may have evolved by descent from the larger antifreeze.

Amino Acid Sequence↗
PubMed2005

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts.

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.

Amino Acid Sequence↗
PubMed2004

Isolation and purification of antifreeze proteins from skin tissues of snailfish, cunner and sea raven.

Antifreeze proteins/polypeptides (AFPs), which are found in diverse species of marine fish, are grouped into four distinct classes (types I-IV). The discovery of skin-specific type I AFPs established that this class contains distinct isoforms, liver-type and skin-type, which are encoded by separate gene families. In this study, type I AFPs were isolated and partially characterized from skin tissues of Atlantic snailfish (Liparis atlanticus) and cunner (Tautogolabrus adspersus). Interestingly, evidence from this study indicates that snailfish type I AFPs synthesized in skin tissues are identical to those circulating in their blood plasma. Furthermore, type II AFPs that are identical to those expressed in liver for export into blood were purified from sea raven (Hemitripterus americanus) skin tissue extracts. It is clear that epithelial tissues are an important source for antifreeze expression to enhance the complement of AFPs that protect fish from freezing in extreme cold environments. In addition, the evidence generated in this study demonstrates that expression of AFPs in fish skin is a widespread phenomenon that is not limited to type I proteins.

Animals↗
PubMed2004

Hyperactive antifreeze protein in a fish.

Fish that live in the polar oceans survive at low temperatures by virtue of 'antifreeze' plasma proteins in the blood that bind to ice crystals and prevent these from growing. However, the antifreeze proteins isolated so far from the winter flounder (Pleuronectes americanus), a common fish in the Northern Hemisphere, are not sufficiently active to protect it from freezing in icy sea water. Here we describe a previously undiscovered antifreeze protein from this flounder that is extremely active (as effective as those found in insects) and which explains the resistance of this fish to freezing in polar and subpolar waters.

Animals↗
PubMed2003

Spatial expression patterns of skin-type antifreeze protein in winter flounder (Pseudopleuronectes americanus) epidermis following metamorphosis.

Two isotypes of Type I antifreeze protein (AFP), the liver-type and the skin-type, have been described from adult winter flounder (Pseudopleuronectes americanus). Although the liver-type AFP has been well studied, the skin-type has just begun to be characterized. It appears to have a wide tissue distribution, be expressed constitutively, and the absence of a signal sequence suggests it is active intracellularly. The current study was designed to examine the onset of skin-type AFP expression during the thickening of the epidermis at metamorphosis from both the nucleic acid and protein levels. The epidermis appeared as a thin layer overlying a thickened dermis at metamorphosis and showed a gradual increase in thickness through the first fall and winter. The onset of skin-type antifreeze expression occurred in conjunction with this epidermal thickening. In situ hybridization and immunohistochemistry showed a distribution of mRNA and skin-type AFP specific for the epidermis and epidermal pavement cells. The AFP immunoproduct showed a distribution intimate with the pavement cell membrane and through the interstitial spaces. This distribution suggests that the AFP may be important in slowing ice crystal formation in these interstitial regions and thus reducing cellular damage due to osmotic imbalance.

Animals↗