Growth Hormone Sequences Research Articles

Epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR: tissue-dependent influence on transcription of the transgenes.

Transgenic mice expressing human urokinase, as well as animals expressing human urokinase receptor under the control of the murine mammary tumor virus (MMTV) long terminal repeat, were established. In the vast majority of the founder animals and their descendants, the transgene was completely methylated, corresponding to down-regulation of transgene expression in the mammary gland. Two lineages with human urokinase receptor as the transgene with mixed methylation of the transgenes were analyzed in more detail. We show here for the first time that the methylation status of the transgene is identical in different organs of an animal, but may differ from animal to animal among the descendents. In the mammary gland, complete methylation of the transgene was incompatible with expression; unmethylated and mixed methylation transgenes gave rise to expression at the RNA as well as at the protein level. The methylation observed was not the consequence of an imprinting process. Surprisingly, in organs other than the mammary gland, such as liver, kidney and spleen, weak expression of the transgene was noted independent of the methylation status of the MMTV promoter. With respect to the molecular mechanism it is unresolved whether the human growth hormone sequence of the transgene harbors a methylation inducing element responsible for the observed methylation pattern.

Follow‐up of antibodies to growth hormone in 210 growth hormone‐deficient children treated with different commercial preparations

The aim of the study was to evaluate the immunogenicity of different commercial recombinant-growth hormone preparations. The presence of antibodies to growth hormone was tested in 210 growth hormone-deficient children at 6-month intervals during treatment for 6-66 months. The patients were treated with three preparations (groups A, B and C of 70 cases each) having the authentic growth hormone sequence. Groups A and B received hormone synthesized by the recombinant DNA technique in E. coli, while the group C preparation was produced in a mammalian cell line. The preparations showed poor immunogenicity and antibodies were found as follows: 1.4% in patients of group A (1 case: binding capacity 0.2 mg/l and Ka 3.5 10(7) l M-1), 2.8% in patients of group B (2 cases; case 1 binding capacity 0.7 mg/l and Ka 1.5 10(7) l M-1; case 2 binding capacity 0.04 mg/l and Ka of 1.8 10(8) and 6.5 10(6) l M-1), and 8.5% in group C (6 cases; binding capacity from 0.4 to less than 0.02 mg/l, Ka from 1.6 10(7) to 3.8 10(8) l M-1). Only two patients of group C presented the antibodies in two subsequent examinations; in the other patients the positivity was found once. In all patients positive samples were found at intervals of 6-24 months after the start of therapy. In all antibody-positive patients growth velocity presented no decrease at the time of antibody detection and was never different to that of negative patients. We conclude that the three commercial preparations examined showed poor immunogenicity without clinical relevance.

Cloning and characterisation of the rabbit growth hormone-encoding gene

The gene encoding growth hormone (GH) has been cloned from a rabbit genomic library, and its sequence has been determined. The rabbit GH gene is similar to other mammalian GH, being comprised of five exons and four introns. As in rodents and artiodactyls, the rabbit GH occurs as a single gene, with no evidence for a cluster of GH-like genes, as is found in primates. The amino acid sequence of rabbit GH is similar to that of pig GH and other conserved mammalian GH, and, like these, differs markedly from the available sequences of ruminant and primate GH. This provides further support for the idea that, in mammals, GH show a slow underlying rate of evolution which has increased markedly on at least two occasions.

Production of germline transgenic Pacific salmonids with dramatically increased growth performance

Transgenic Pacific salmon have been produced by microinjection of a DNA construct consisting of chinook salmon (Oncorhynchus tshawytscha) growth hormone sequences driven by an ocean pout (Macrozoarces americanus) antifreeze protein promoter. This construct was retained in approximately 4% of fish derived from injected eggs, and resulted in dramatic enhancement of growth relative to controls. For coho salmon (O. kisutch) at 15 months of age, the average size of transgenic fish was more than 10-fold that of controls, with the largest fish more than 30-fold larger than nontransgenic siblings. Dramatic growth enhancement was also observed in transgenic rainbow trout (O. mykiss), cutthroat trout (O. clarki), and chinook salmon using this same gene construct. Transgenic coho salmon underwent precocious parr–smolt transformation during their first fall, approximately 6 months in advance of their nontransgenic siblings. At 2 years of age, five male transgenic coho salmon became sexually mature, and four of these transmitted the gene construct to sperm, the negative fish being transgenic in blood but not fin tissue. These results show that while some fish are mosaic for the gene construct in different tissues, most are transgenic in both germline and somatic tissue.

Obtaining A More Resolute Teleost Growth Hormone Phylogeny by the Introduction of Gaps in Sequence Alignment

In order to obtain a more resolute phylogeny of teleosts based on growth hormone (GH) sequences, phylogenetic analyses were performed in which deletions (gaps), which appear to be order specific, were upheld to maintain GH′s structural information. Sequences were analyzed at 194 amino acid positions. In addition, the two closest genealogically related groups to the teleosts, Amia calva and Acipenser guldenstadti, were used as outgroups. Modified sequence alignments were also analyzed to determine clade stability. Analyses indicated, in the most parsimonious cladogram, that molecular and morphological relationships for the orders of fishes are congruent. With GH molecular sequence data it was possible to resolve all clades at the familial level. Analyses of the primary sequence data indicate that: (a) the halecomorphean and chondrostean GH sequences are the appropriate outgroups for generating the most parsimonious cladogram for teleosts; (b) proper alignment of teleost GH sequence by the inclusion of gaps is necessary for resolution of the Percomorpha; and (c) removal of sequence information by deleting improperly aligned sequence decreases the phylogenetic signal obtained.

Complete Amino Acid Sequence of Crocodile Growth Hormone

This paper describes the isolation and complete amino acid sequence of growth hormone (GH) from the pituitary gland of the crocodile, Crocodylus novaeguineae. GH was detected in an alkaline extract of the pituitary by specific immunoblot reactivity with rabbit antisera against cattle and salmon GH and isolated in pure form by DEAE-cellulose chromatography and by reversed-phase high-performance liquid chromatography. The yield of the GH was 2.2 mg from 3.5 g of wet tissue. Crocodile GH consists of 190 residues and is highly similar to chicken, duck, and sea turtle GHs with 90-92% structural similarity, although it shows only 27% identity with crocodile prolactin. A molecular phylogenetic tree of GH from 39 vertebrate species is proposed on the basis of sequence comparison.

The effect of changes in nucleotide sequence coding for the N-terminus on expression levels of ovine growth hormone variants in Escherichia coli

The expression levels of coding sequences for pituitary growth hormone, introduced into Escherichia coli by genetic manipulation techniques, vary markedly according to the precise sequence introduced. In order to understand the basis of this variation more fully, we have studied the relationship between the level of expression in E. coli of a series of ovine growth hormone variants and the nucleotide sequences coding for their N-terminal regions. Sequence variation resulted from the introduction of deletions, or site-directed mutations, into a plasmid containing the coding sequence for ovine growth hormone preceded by the initiation codon and 25 bases derived from β-galactosidase or linker regions of plasmid pUC8. The expression levels of the variants varied from less than 0.01% to over 34% of the total cell protein, indicating that changes in the nucleotide sequence close to the initiation codon had a marked effect on expression level. The results of a comparison of closely related sequences in pairs of plasmids giving poor or good expression are consistent with the hypothesis that poor translation of growth hormone mRNAs is caused by the presence of secondary structures close to the initiation codon. Secondary structures are identified that appear to explain the variation in expression levels.

Cloning and nucleotide sequencing of rat lymphocyte growth hormone cDNA.

The messenger RNA for rat growth hormone (GH) was isolated from rat spleen lymphocytes and cloned as a cDNA after PCR. Four clones, including part of the prehormone sequence and the full-length mature GH sequence, were obtained and sequenced. The sequence data revealed that rat lymphocyte GH was identical to that reported for pituitary GH. The results do not support the expression by alternate splicing of the 20-kD variant described in the pituitary. We also cloned the promoter of lymphocyte GH and analyzed its nucleotide sequence including 300 base pairs of the 5'-flanking region. The promoter sequence we obtained did not exactly match that reported in the literature because of reading compressions on the gel. However, parellel sequencing of thymus, spleen and pituitary GH promoter sequences gave identical patterns of compressions in the gel. The results suggest that the sequence in all the tissues was the same. Collectively, these studies demonstrate that lymphocytes express authentic GH mRNA and suggest that GH gene expression may be regulated in immune tissue by similar transcriptional factors to those described in the pituitary.

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

AUUUA Is Not Sufficient To Promote Poly(A) Shortening and Degradation of an mRNA: the Functional Sequence within AU-Rich Elements May Be UUAUUUA(U/A)(U/A)

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

Pattern of nucleotide substitutions in growth hormone-prolactin gene family: a paradigm for evolution by gene duplication.

The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among members of duplicated gene families and among species was examined by using reported gene sequences of growth hormone, prolactin and their receptors. Sequence divergence among species was found to show a general tendency in which a generation-time effect is pronounced for synonymous substitutions but not so for nonsynonymous substitutions. Divergence among duplicated genes is characterized by the relatively high rate of nonsynonymous substitutions, i.e., the rate is close to that of synonymous ones. In view of the stage- and tissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian selection.

Introduction and expression of the bacterial genes cysE and cysK in eukaryotic cells

The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.

Procedural Protocol, Survival to Hatching and Plasmid DNA Fate After Microinjection into Tilapia Zygotes

The plasmid vector, pBRd-AK1-BGH4.6.10 (pBGH), containing the bovine growth hormone sequence driven by an avian retroviral long terminal repeat (LTR) was microinjected at 0.1, 0.5, 1 or 5 ng of pDNA/20 nl of physiological saline into tilapia, Oreochromis mossambicus × O. niloticus embryos. In 24 replicates, normally 60 zygotes were microinjected with either the entire linear 8.5 kb pBGH/ClaI vector or a 3.8 kb pBGH/SalI restriction fragment. Overall mean survival to fry hatching was 7.6% in plasmid DNA-microinjected, 15.6% in sham-microinjected and 48.1% in uninjected control embryos. There were no significant (P > 0.05) differences in survival to hatching between those embryos microinjected with the 3.8 or the 8.5 kb restriction fragment, nor was there a trend toward decreasing survival as the plasmid DNA concentration increased from 0.1 to 5 ng. The significant (P < 0.05) increase in survival among uninjected control embryos to hatching indicates that microinjection trauma was the major cause of mortality. Large quantities of plasmid DNA were recovered from pooled-embryo samples. Multiple bands (positive signals) were detected usually in the high molecular weight (HMW) genomic DNA regions. Position shifting of these HMW bands upon digesting with various restriction endonucleases provided evidence for plasmid DNA integration into tilapia embryo chromosomal DNA. Otherwise, these positive signah may have been end-twnd ligations of increasingly longer plasmid DNA constructs. Putative transgenic O. mossmbicus × O. niloticus were found among eight of 27 surviving adults.

The complete nucleotide sequence of the Atlantic salmon growth hormone I gene

Two closely related genes encoding growth hormone were isolated from Atlantic salmon by genomic cloning. From one of these genes a total of 6500 nucleotides were determined including 3900 nucleotides in exons and introns and about 600 and 2000 nucleotides in 5' and 3' flanking regions. The gene is organized in six exons and encodes a polypeptide of 210 amino acids including a 22 amino acids signal sequence. The promoter region contains a typical TATA box 21 nucleotides upstream from the transcription start site. At the 3' end, three putative poly(A) signal sequences are present. The last two are within a 121 nt inverted repeat.

Involvement of zinc in binding of hGH to the prolactin receptor

This chapter describes the species specificity and structure–function relationships of growth hormone (GH). GH is a protein hormone derived from the anterior pituitary gland. It comprises a single polypeptide chain of about 190 amino acids, containing two intrachain disulfide bridges. The apparent rapid evolution of GH in the primates makes sequence studies on GH for primates other than man of particular interest. The sequence of GH from the rhesus monkey has been reported. This is very similar to that of human GH, differing at only four residues, suggesting that the rate or evolution of GH because the divergence of old-world monkeys and great apes has been slow. The implication is that GH underwent a period of remarkably rapid evolution early in primate evolution. An alternative explanation is that the primate and nonprimate GH genes are not strictly homologous, implying that a second gene is present in the human genome, homologous with nonprimate GHs and a second gene is present in the nonprimate mammalian genome, homologous with human GH. The situation is complicated by the occurrence of a cluster of GH-like genes in man.

Isolation of sockeye salmon growth hormone utilizing serum triiodothyronine enhancement in rainbow trout to monitor biological activity

Growth hormone (GH) was isolated from sockeye salmon ( Oncorhynchus nerka) pituitary glands using established techniques of affinity and gel filtration chromatography, and preparative polyacrylamide gel electrophoresis. The GH activity was followed throughout the fractionation procedure with a bioassay based on the increase of serum triiodothyronine (T 3) in rainbow trout ( Oncorhynchus mykiss), measured by radioimmunoassay (RIA). Aminoterminal amino acid sequence analysis and subsequent comparison with established GH sequences from other Oncorhynchus sp. were used to confirm the isolation of sockeye salmon GH (ssGH). The bioassay was sensitive to a dose of 55 ng of purified ssGH/g fish. Monomeric GHs, located in the carbohydrate-poor protein fraction, were the only pituitary components that elevated serum T 3. Twenty-four hours after GH injection was an appropriate and practical time to blood sample, allowing completion of the bioassay, including RIA, in 3 days. The generic homology, between the source of pituitaries and the bioassay animals used in this study, should permit the bioassay to be useful during GH isolation from pituitaries of all Oncorhynchus sp.

Cloning and sequencing of the gilthead seabream ( Sparus aurata) growth hormone-encoding cDNA

The cDNA clones encoding gilthead seabream (gsb) ( Sparus aurata) growth hormone (GH) have been isolated from a cDNA library prepared from seabream pituitary gland poly (A) +RNA. The cDNA library was screened using red seabream and rainbow trout GH cDNAs. The complete nucleotide (nt) sequence of gsb GH has been determined. The cDNA sequence codes for a polypeptide of 204 amino acids (aa), including a putative signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 55 and 236 nt long, respectively. The predicted aa sequence of gsbGH revealed 97%, homology with red seabream GH, 95%, with tuna GH, 85% with yellowtail GH, and 65% with rainbow trout GH.

Amino acid sequence of growth hormone isolated from medium of incubated pituitary glands of tilapia ( Oreochromis mossambicus)

The amino acid sequence of tilapia ( Oreochromis mossambicus) growth hormone (GH) was determined directly by Edman degradation of peptide fragments generated by lysyl endopeptidase and trypsin digestion. The N-terminal residue was deduced to be pyroglutamic acid through the use of pyroglutamyl aminopeptidase; its removal allowed amino acid sequence determination of the remainder of the N-terminal trypsin peptide by Edman degradation. Tilapia GH is composed of 187 amino acid residues and shows high similarity to other perciform GHs. Sequence identities are: 89% with tuna GH, 83% with bonito GH, 85% with yellowtail GH, 89% with red sea bream GH, and 34% with bovine GH. The two asparagine residues (Asn-148 and Asn-184) were recovered by Edman degradation, suggesting the absence of N-glycosylation.

The complete amino acid sequences to two variants of growth hormone from Atlantic cod (Gadus morhua)

The complete amino acid sequences of two variants of cod growth hormone (GH) have been determined. The GHs, which have apparent molecular weights of 20K and 22K in SDS-PAGE, consist of 185 amino acids and have calculated molecular weights of 20,733 and 20,805, respectively. Comparison of the two sequences showed only one amino acid difference between the variants, with Lys at position 151 in the 22K GH changed to Gly in the 20K GH. The substitution of a charged amino acid by one which contains no sidechain might be expected to be reflected in the isoelectric point of the molecule. However, the observed pI for both the 20K and 22K GHs was 5.8. The difference in apparent molecular weights by SDS-PAGE suggests the existence of a conformational difference between the variants which is attributable to the observed substitution. This conclusion is in agreement with our previous data obtained from radioimmunoassay studies where the 20K GH shows only 25% cross-reactivity in an assay developed for the 22K GH. Alignment of the cod GH sequence with those of other teleost GHs reveals cod GH to be most similar to advanced marine fish such as tuna, sea bream, bonito, and yellowtail (76-83% identity), whereas it is 62-66% identical to flounder and chum salmon GH.

Identification of a DNA-binding site for the transcription factor GC2 in the promoter region of the p12 gene and repression of its positive activity by upstream negative regulatory elements.

Mouse secretory protease inhibitor p12 is significantly transcribed by the cells from the seminal vesicle, the coagulating gland, the ventral prostate, and to a lesser level by the pancreas. It is otherwise undetectable in every other tissue examined. To study the molecular mechanisms involved in this model of cell-specific control of gene expression, we cloned fragments containing various lengths of the p12 promoter upstream of the CAT reporter gene. We demonstrated that p12 sequences from +34 to -108 relative to the CAP site can confer a constitutive level of CAT expression following transient transfection in non-prostatic CV1 and GH4C1 cells. We identified within this minimal p12 promoter the cis-acting sequences needed to direct such a significant level of CAT expression. A DNA-binding site (p12.A) highly homologous to the rat growth hormone (rGH) sequence recognized by the trans-acting factor GC2 was identified between the TATA- and the double CAAT-box sequences from the p12 promoter. Using competition and mutation analysis, we provide evidence that the positively acting p12.A-binding protein is likely to be the rGH GC2 transcription factor, suggesting that the same, or a very similar factor, regulates expression of both rGH and p12 genes. By further analysis of the p12 5'-flanking sequences, we demonstrated that plasmids including sequences from -109 to -843 can strongly repress the level of transcription directed by this minimal p12 promoter, providing evidence for the presence of cis-acting negative regulatory elements critical for the establishment of p12 gene extinction in non-prostatic cells.