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C L Hew

Publications and source records attributed to C L Hew.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1995

Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish.

Zebrafish embryos have three or four identifiable primary motoneurons per hemisegment. We previously reported that, while several ventral cells initially express the zebrafish Islet-1 (Isl-1) gene, a member of the LIM/homeobox gene family, the expression of this gene becomes restricted to a single or a pair of cells slightly anterior to each segment border by 16 hr after fertilization. Double staining by in situ hybridization and immunohistochemistry strongly suggested that these cells were mainly rostral primary motoneurons. Here, we have isolated two novel zebrafish cDNA clones for more Isl-1 family genes, termed zfIsl-2 and zfIsl-3. zfIsl-2 mRNA starts to be expressed in the ventral midsegmental cells per hemisegment around 15 hr. Double labeling experiments have shown that these midsegmental cells are the caudal primary motoneuron (CaP) and its variant equivalence pair. Our results revealed the heterogeneity in the expressed genes among primary motoneurons before the fates of the primary motoneurons are irreversibly determined, and further suggest the involvement of the Isl-1 and zfIsl-2 genes in the determination of cellular identities by primary motoneurons in embryonic zebrafish. zfIsl-3 mRNA is not expressed in motoneurons but is expressed at 17 hr, mainly in the ventral myotomes. This suggests that zfIsl-3 may be involved in the regional specification of the myotome and also in target recognition by CaP. zfIsl-2 is also expressed throughout the developing eye and tectal region of the midbrain, the target for the retinal axons. In the ventral spinal cord of the spadetail mutant embryo, which has defects in the somites, the cells expressing zfIsl-2 mRNA significantly decreased in number in contrast to the increase in cells expressing Isl-1 mRNA, suggesting the influence of the somites on the expression of both genes.

Amino Acid Sequence↗
PubMed1995

Functional analysis of estrogen-responsive elements in chinook salmon (Oncorhynchus tschawytscha) gonadotropin II beta subunit gene.

The salmon gonadotropin II gene regulates ovulation and spawning. Analysis of the 5' flanking sequence of the hormone-specific beta-subunit of salmon gonadotropin II (sGTHII beta) gene reveals the presence of several presumptive estrogen-responsive elements (ERE). The participation of ERE in the control of sGTHII beta gene transcription was examined by the transient expression of sGTHII beta gene promoter-chloramphenicol acetyltransferase chimeric DNA constructs in HeLa cells, with the cotransfection of a rainbow trout estrogen receptor expression vector. Three ERE have been identified: the proximal ERE [pERE, at -267 base pair (bp) from the transcription start site], the distal ERE (dERE, at -2698 bp, three GGTCA motifs each separated by exactly 31 bp), and the half-ERE (1/2ERE, at -157 bp as a GGTCA motif), respectively. The pERE (TGTCAATCTGACC) represents a novel but less effective variation of the consensus ERE (cERE). The dERE is a unique estrogen-induced enhancer. It requires the participation of the pERE to be functional and the enhancer activity of pERE and dERE is promoter specific. The contribution of 1/2ERE is minor and is not cell-type specific. The activation of the ERE in the sGTHII beta gene and the synergistic cooperation between the dERE and pERE by estradiol-17 beta is dose dependent. DNA sequences in the vicinity of the ERE decreases their hormone responsiveness and the synergism between dERE and pERE. These negative regions may contribute to the quiescent endocrine state of the sGTHII beta gene during the regressive phase of the reproductive cycle in teleost.

Animals↗
PubMed1995

A gene encoding chinook salmon (Oncorhynchus tschawytscha) gonadotropin alpha subunit: gene structure and promoter analysis in primary pituitary cells.

The cDNA clones coding for the chinook salmon gonadotropin alpha (sGTH alpha) subunit were isolated from a salmon pituitary cDNA library. The full-sized cDNA clone, sGTH alpha-C2, was 678 bp in size and encoded a precursor polypeptide of 114 amino acids, including a signal peptide of 22 amino acids. The deduced mature sGTH alpha subunit polypeptide is identical with the major alpha subunit of chum salmon gonadotropin. Genomic clones coding for the sGTH alpha subunit were isolated from a partial BamHI genomic library in lambda EMBL3 using the cDNA as a probe. Seven positive clones were identified from 1 x 10(6) plaques. Restriction mapping of positive clones suggested that there were two copies of the GTH alpha subunit gene in the salmon genome. The 2 kb of 5'-flanking region and the 1.1 kb of the entire sGTH alpha subunit coding region were sequenced from the genomic clone, sGTH alpha-G1. The sGTH alpha subunit gene contains four exons and three introns. However, its introns are smaller than those in the mammalian hormones. Inspection of the sGTH alpha subunit promoter region revealed several potential cis-acting elements, including the gonadotrope-specific element (GSE), Ap-1, AP-2, and others. Functional analysis of the sGTH alpha subunit promoter by the transient transfection of several sGTH alpha/CAT chimeric plasmids into rainbow trout pituitary cells suggests that its pituitary-specific expression is GSE-dependent. The activity of the chimeric plasmids can be stimulated by 8-bromo-cAMP. These experiments indicate that pituitary primary cells can be used to dissect various cis-acting elements important for salmon GTH alpha subunit gene expression.

Amino Acid Sequence↗
PubMed1995

Expression of the antifreeze protein gene in transgenic goldfish (Carassius auratus) and its implication in cold adaptation.

Ocean pout (Macrozoarces americanus) antifreeze protein (AFP) genes (approximately 10(6) copies) were microinjected into the oocytes of goldfish (Carassius auratus). Out of a total of 303 oocytes injected, 235 (77.6%) were normally fertilized after in vitro maturation and insemination, and 136 (57.9%) of the fertilized eggs were hatched. Dot blot analysis of genomic DNA from 2-month-old fish indicated that 30 of the fish (22%) were positive for the AFP transgene. The numbers of integrated AFP genes ranged from a single copy to multiple copies per cell. Two P1 male founders, Y45 and GV16, were crossed separately with a control female, and the inheritance of the AFP transgene was analyzed. Results from Southern blotting and polymerase chain reaction were found to be 7% (5 out of 82) and 58.8% (20 out of 34), respectively, indicating that these two founders are mosaic. The percentage of positive F2 generated from Y45F1 crossed with a control female was 56%, consistent with a Mendelian inheritance. Mature AFP detected by immunoblotting was expressed in both F1 and F2 offspring. These studies indicate that the antifreeze protein gene is successfully transferred and expressed in goldfish. Transgenic goldfish are significantly more cold tolerant than controls when challenged with low temperatures. These studies suggest that the AFP gene may have application in providing cold tolerance in addition to freeze resistance for a variety of fish species.

Acclimatization↗
PubMed1994

Zinc and DNA binding properties of a novel LIM homeodomain protein Isl-2.

LIM homeodomain proteins are a family of recently characterized proteins which contain, in addition to a homeodomain, two tandem repeats of conserved Cys-His motifs termed as LIM domains. We have recently isolated several clones from a chinook salmon pituitary cDNA library that encode two novel LIM homeodomain proteins, Isl-2 and Isl-3, which are structurally related to rat Isl-1. In the present study, we used the salmon Isl-2 to determine the role of LIM domains in DNA binding. Several glutathione S-transferase (GST) fusion proteins containing either full length Isl-2 or various portions of this protein were expressed in bacteria. Zinc blot analysis reveals that the LIM domains produced in bacteria are capable of binding zinc. Gel shift analysis indicates that all homeodomain-containing fusion proteins are able to bind to a TAAT target sequence while the fusion proteins containing only the LIM domain are not. In contrast to a previous observation that the LIM domains of rat Isl-1 have an inhibitory role in DNA binding, full length salmon Isl-2 containing both the LIM domains and a homeodomain can bind to a TAAT target sequence. To further examine the role of LIM domains in DNA binding, several GST fusion proteins were used to select specific target DNA sequences from a pool of randomly incorporated oligonucleotides. Specific target DNAs were selected by fusion proteins containing the homeodomain or the full length Isl-2, but not by LIM domain only fusion proteins, indicating that the LIM domain alone is not involved in DNA binding. The selected target DNAs were cloned and sequenced. They revealed two classes of consensus, C/TTAATG/TG/A and C/TTAAGTG, for both the homeodomain and full length Isl-2. The two classes of consensus competed with each other for binding to the homeodomain. The equilibrium dissociation constants for DNA binding, estimated by Scatchard analysis, were similar for the homeodomain and full length Isl-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Single crystals of a type III antifreeze polypeptide from ocean pout.

Antifreeze protein (HPLC-2) from ocean pout was purified from serum using column chromatography on Sephadex G75 and reverse-phase HPLC columns. Single crystals were grown by batch methods at 4 degrees C from a 1.5 M solution of ammonium sulphate (pH 7.1). The crystals diffracted to about 2.5 A resolution at 4 degrees C and belong to the monoclinic space group P2(1), with cell parameters: a = 39.77 A, b = 58.51 A, c = 30.27 A, beta = 102.28 degrees, with two molecules of 6000 M(r) per asymmetric unit.

Animals↗
PubMed1994

The chinook salmon gonadotropin II beta subunit gene contains a strong minimal promoter with a proximal negative element.

The salmon pituitary expresses two distinct gonadotropins, gonadotropin I (GTHI) and gonadotropin II (GTHII). These two hormones are synthesized in distinct pituitary cells and secreted at different stages during the reproductive cycle. To study the transcriptional regulation of the hormone-specific beta-subunit of GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the 5'-flanking region was characterized and sequenced. The pituitary specificity of sGTHII beta was examined by analyzing sGTHII beta promoter activity in homologous primary pituitary cells derived from spawning rainbow trout and in a collection of heterologous cell lines. Various lengths of the 5'-flanking region of the sGTHII beta gene were ligated into a vector encoding the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting sGTHII beta/CAT chimeric constructs were analyzed using transient expression systems. Several constructs (-3500CAT, -1260CAT, -563CAT, and -39CAT) displayed readily detectable CAT activity in the pituitary cells derived from spawning male and female rainbow trout. In contrast, three of the constructs (-3500CAT, -1260CAT, and -563CAT) were expressed only at background levels in a variety of heterologous cell lines, suggesting that the 5'-flanking sequence of sGTHII beta contains information dictating its pituitary specificity. A silencer sequence (-95 to -35, pSil) was identified, which might function to repress sGTHII beta gene expression in the nongonadotropes or in the gonadotropes at developmental stages that precede final maturation and spawning.

Animals↗
PubMed1994

Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin II beta subunit gene during salmon maturation.

The role of testosterone (T) and 17 beta-estradiol (E2) in the control of chinook salmon gonadotropin II beta subunit (sGTHII beta) gene was examined. Both E2 and T specifically stimulated GTHII beta gene expression in cultured juvenile rainbow trout pituitary cells. 5'-Flanking regions of the sGTHII beta gene linked to the chloramphenicol acetyltransferase (CAT) expression vector were transfected into these pituitary cells, and cultures were treated with steroid hormones. Estrogen-stimulated CAT activity occurred with constructs containing a 13-base pair estrogen responsive element (ERE) sequence [proximal ERE (pERE)] located at -273 to -260 upstream of its transcriptional start site. Binding specificity of pERE was confirmed by mobility shift and DNA methylation interference assays using the DNA binding domain of the human estrogen receptor. Interestingly, the pERE functioned to derepress the activity of the proximal silencer (pSil) only in the pituitary cells of juvenile trout but not in cells derived from maturing and sexually matured fish. Another potential ERE sequence comprised of three tandemly linked half-ERE palindromes was located from -2736 to -2659 [distal ERE (dERE)] of the sGTHII beta gene. Distal ERE might be responsible for the steroid responsiveness of the longest sGTHII beta/CAT construct (-3500CAT) observed in the pituitary cells of maturing fish. The function of pERE and dERE were further examined in the heterologous HeLa cells by mutagenesis and cotransfection with a rainbow trout estrogen receptor expression vector. Disruption of the palindromic structure of pERE severely impaired its function. When the sequences between pERE and dERE were deleted, a 200-fold increase in CAT activity was observed in response to E2. A model is proposed to describe the regulation of GTHII beta gene expression at different reproductive stages.

Animals↗
PubMed1994

Bulk isolation and identification of fish genes by cDNA clone tagging.

The fish has long been a popular research model for various disciplines in the life sciences, but its molecular biology remains largely unexplored, and the number of fish genes identified at present is still limited. To increase the repertoire of fish genes to facilitate further research, we have embarked on cDNA clone tagging studies in several teleosts. The basic procedure is partial sequencing of randomly selected cDNA clones by a single sequencing reaction to obtain nucleotide sequences of about 200 to 300 bp for each clone. These partial DNA sequences can be used as tags to identify fish homologous genes by searching for sequence similarity in DNA databases. By this method, we have sequenced a total of 265 clones from pituitary and liver cDNA libraries from five species of fish and identified 83 clones encoding 55 different genes. These 55 genes are described for the first time in their species, and 37 of them are described for the first time in fish. These preliminary data indicate that cDNA clone tagging is useful for rapidly increasing the number and availability of genes in teleosts. Several applications of these tagged random clones are discussed.

Animals↗
PubMed1993

Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): presence of two functional genes, GH-I and GH-II, and a male-specific pseudogene, GH-psi.

Two chinook salmon (Oncorhynchus tshawytscha) growth hormone genes (a functional GH-I gene and a pseudogene, GH-psi) were isolated and characterized. The GH-I gene sequence consists of 1.9 kb of 5'-flanking sequence, 4.1 kb of transcribed region, and 64 bp of 3'-flanking sequence, and contains 6 exons and 5 introns. The pseudogene, GH-psi, spanning 4.1 kb, has a similar structure as the GH-I gene. However, it has one wrong splicing sequence at the intron 1/exon 2 junction, one premature termination codon in exon 5, and a deletion in the last half of exon 5 and the first part of intron 5. In addition to GH-I gene and GH-psi, a third GH gene, GH-II, was identified by the polymerase chain reaction (PCR) and subsequently shown to be the second functional GH-II gene. To study the linkage arrangement of these three GH genes, 50 unrelated chinook salmon (25 males and 25 females) and one chinook salmon family were analyzed by PCR. The results showed that GH-psi exists only in males and that it segregates from father to sons. These results suggest that GH-psi is sex specific and probably resides on the Y chromosome. Together these results indicate that there are three GH genes in the genome of male chinook salmon, and only two GH genes in the females. The extra GH gene in the male is, however, a pseudogene.

Amino Acid Sequence↗
PubMed1992

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.

Amino Acid Sequence↗
PubMed1992

Protein interaction with ice.

Many organisms have evolved novel mechanisms to minimize freezing injury due to extracellular ice formation. This article reviews our present knowledge on the structure and mode of action of two types of proteins capable of ice interaction. The antifreeze proteins inhibit ice crystal formation and alter ice growth habits. The ice nucleation proteins, on the other hand, provide a proper template to stimulate ice growth. The potential applications of these proteins in different industries are discussed.

Antifreeze Proteins↗
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

Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A gene encoding chinook salmon (Oncorhynchus tschawytscha) prolactin: gene structure and potential cis-acting regulatory elements.

A full-length chinook salmon (Oncorhynchus tschawytscha) prolactin (PRL) gene, the first genomic clone of a teleost prolactin, was isolated and fully sequenced. The chinook PRL genomic sequence spans 6.4 Kb, including 2.4 Kb of 5' flanking sequence, 3.0 Kb representing the five exons and four introns of the complete PRL gene, and 0.9 Kb of 3' flanking sequence. The transcriptional start site of the PRL gene was mapped through the agreement of both primer extension and S1 nuclease protection assay. The 5' flanking region of the PRL gene was searched for potential cis-acting elements based on the consensus binding site of trans-acting factor Pit-1, known to be involved in PRL gene expression in mammals. Functional analysis of PRL promoter by the transient transfection of several PRL promoter/CAT chimeric plasmids into rainbow trout pituitary cells suggests a functional PRL promoter whose cell-specific activity is most likely governed by both positive and negative mechanisms.

Amino Acid Sequence↗