Growth Hormone Gene Sequences Research Articles

Cloning and expression of the growth hormone gene and its effect on the proliferation of skeletal muscle cells of Guizhou cattle

The aim of this study was to investigate the expression of growth hormone (GH) gene on skeletal muscle cell proliferation of Guizhou cattle. The coding sequence of cattle GH gene was amplified by reverse transcription PCR, cloned into the pUCM-T vector and then used to construct the GH gene overexpression vector pEGFP-N3-GH. The expression of the GH gene in skeletal muscle-related tissues (psoas major and longissimus dorsi) of Guizhou cattle was determined by real-time fluorescent quantitative PCR (RT-qPCR). This was followed by culturing and identification of the bovine primary skeletal muscle cells. Subsequently, we introduced the GH gene overexpression vector into the cells to investigate its effect on the proliferation of bovine skeletal muscle cells and the expression of insulin like growth factor 1 and 2 genes related to skeletal muscle growth and development. RT-qPCR results showed that the expression level of GH gene was higher in the psoas major than in the longissimus dorsi of Guizhou cattle, and the expression level in the psoas major of Guanling cattle and Weining cattle was significantly higher than in the longissimus dorsi (P<0.05). The transfection and proliferation results showed that pEGFP-N3-GH significantly increased the expression of GH, IGF-1, and IGF-2 genes in skeletal muscle cells compared to pEGFP-N3 (PP<0.05), and that overexpression of the GH gene also significantly increased the proliferation rate of skeletal muscle cells at the four periods examined (PP<0.01). Our results suggest that GH gene can promote the proliferation of skeletal muscle cells of Guizhou cattle and exerts a positive regulatory effect. This lays the foundation for further exploring the mechanism by which the GH gene affects the growth and development of Guizhou cattle.

Molecular Characterisation Of Growth Hormone (Gh) Gene In Indian Dromedary And Bactrian Camel

Molecular characterisation of 613 bp long growth hormone (GH) gene fragment spanning partial exon-1, intron-1, exon-2 and partial intron-2 was done in Indian (one humped) dromedary and Indian (double humped) bactrian camel through PCR amplification, sequencing and bioinformatics analysis. The sequence variations within and between single and double humped Indian camels were observed. In the Indian single humped camels of Mewari, Kachchhi and Bikaneri breed at position 264 C>T variation was seen. At this particular locus animals with single and double peaks in the sequence chromatograms were observed. Accordingly, two allele (C, T) and three genotype (CC, CT, and TT) were identified in the Indian dromedary camels. In Indian Bactrian camel at position 264 only C allele and CC genotype was identified. In double humped camel at position 242, A>G and at position 469 G>A transition variation was observed compared to single humped camel. The different sequences obtained were submitted to NCBI and Gen Bank Accession No. MT478653 for C allele Indian Dromedary, MT478654 for T allele Indian Dromedary and MT478655 for Indian Bactrian camel were obtained. Similarity ranging from 98% to 100 % was observed with available GH Sequences of camel at GenBank. Camel sequence was found to have close similarity with other Camelidae family members like Lamapacos (97.07%) and Lamaglama (96.58%). The evolutionary relationship between sequences showed close relationship between dromedary and bactrian camel followed by vicugna and llama. The domesticated species like cattle, buffalo, sheep, goat, yak and mithun were distantly related to camel. The present study showed close similarity between GH gene sequence pattern of Indian one and double humped camel except transition variation at two positions in bactrian camel.

Growth Hormone Gene Polymorphisms in Mature Healthy Male Muscovy Ducks in Egypt

Blood samples were obtained from 28 mature healthy male Muscovy ducks for screening of a partial sequence of Growth hormone gene (GH gene) for single nucleotide polymorphisms using PCR amplification of the extracted DNA. The results of our research revealed one locus of exon 2 of growth hormone gene and its partial flanking intron. Polymorphisms in this locus were investigated by using direct sequencing and single strand conformational polymorphism (SSCP) technique. One unique pattern of SSCP was detected. Nucleotide sequencing was performed to endorse the current result which proved the absence of single nucleotides' polymorphisms (SNPs) or any other type of polymorphisms in the studied locus. The DNA sequencing and PCR-SSCP patterns confirmed that there was no polymorphism in all studied birds. In turn, it is spelled that PCR-SSCP failed to produce electrophoresis patterns capable of discriminating between the GH gene loci in Muscovy duck breed revealing no polymorphism in all studied individuals. Also, we compared our sequences with the published sequence on the gene bank of GH gene in Mallard ducks under the following accession number AB158762.1. Three SNPs were detected between Muscovy and Mallard breeds which are (94 A-G, 271 C-T and 298 G-A) these SNPs led to change of two amino acids proline and valine amino acids in GH protein in mallard ducks into serine and isoleucine in Muscovy ducks. In conclusion, these changes in the nucleotide sequence, amino acid and protein chain may be the main cause of differences of the phenotypic features, productive and reproductive traits between Muscovy and mallard duck breeds.

English

Many factors contribute to the underdevelopment of oceanic aquaculture. The most significant factor of the development of hybrid grouper culture is the supply of satisfactory quantities of fast growing juveniles to stock grow-out systems at a minimized cost. In order to evaluate the growth hormone status of hybrid grouper (Epinephelus fuscoguttatus ♀&times; Epinephelus polyphekadion ♂), cDNA encoding growth hormone from the liver of juvenile fish was cloned and characterized. qPCR method was used to determine the expression of the growth hormone in the tissues. Relative expression of growth hormone in the brain was found to be highest (290.91 folds) and lowest in the stomach (1.35 folds). Bioinformatics tools were used to analyze the growth hormone proteins and prediction of its physicochemical properties, and amino acid compositions, as well as Gene Ontology, GO term predictions. The cloned hybrid grouper growth hormone gene sequence was found to be clustered monophyletically with solid bootstrap backing. It was found to be close to the Cromileptes altivelis growth hormone gene sequence and is positioned in the identical clade and consequential from the identical family. The expression pattern is comparable to that comprehended in other fishes and provides extra data for molecular biological studies on hybrid grouper fish. Key words: Growth hormone, bioinformatics analysis, tissue expression, gene ontology, hybrid grouper &nbsp;

Genetic Polymorphisms in Exon-3 region of Growth Hormone Gene in the Egyptian Goat Breeds

Background: In Egypt, the importance of goats comes from its potential source of meat and milk production. Thus, goat productivity improvement acts as a global strategy to provide Egyptians by important source of protein feeding. Molecular techniques have been employed to analyze growth hormone (GH) gene that promotes goat muscles, bone formation, regulating fat content and other important traits in goat. Our objective was to document and find out an accurate genetic marker sites, single nucleotide polymorphisms (SNPs) in exon-3 region of GH gene in some Egyptian goat breeds. Methods: Fifteen blood samples were collected from three Egyptian goat breeds (Baladi, Barki and Zaraibi), DNA was isolated and primer was used to amplify GH gene region corresponding to exon-3 region. Finally, sequencing methodology was applied for PCR products regarding the three goat breeds. Results: The results showed four nucleotide substitutions; The first SNP (CaT) was located at nucleotide position 106, the second SNP( GaA) was located at nucleotide position118 and the third SNP (GaA) was located at nucleotide position 128 .Finally, the fourth SNP (TaC) was located at nucleotide position 136. It was found that the SNP (GaA) at nucleotide position 128 caused an amino acid change from Glycine to Serine in the protein sequence of GH gene. Conclusion: Our findings demonstrated several genetic variations across exon-3 region of GH gene which can be used as marker in selection of goats with high valued traits.

Sequencing of growth hormone gene for detection of polymorphisms and their relationship with body weight in Harri sheep

This study aimed to evaluate the relationship between growth hormone (GH) gene polymorphism and estimated body weight in Harri sheep. One hundred Harri sheep lambs were used to determine the birth weight (BW) and weight at 120 days of age. The daily live-weight gain (DLWG) 0-120 days (g) was calculated. The complete CDS of the Harri sheep GH gene is 2117pb in length (GenBank acc. no. KU255857). Three novel SNPs were detected by comparing with GenBanke acc. no. X12546_1. The G871A SNP in intron II, G1383A in exon IV that resulted in conversion of the amino acid arginine number 121 to lysine (R121K) and the A1509G in intron IV. Each SNP was found on both alleles the mutant homozygote was more common (48, 56 and 50%) than the heterozygote (30, 18 and 20%) for G871A, G1383A and A1509G respectively.A positive significant (P is less than 0.05) correlation between growth traits (BW, 120 day body weight and DLWG) and SNP and a highly significant correlation with the genotype were detected. The regression analysis indicated the positive effect of genotype and SNPs on the growth traits. Individuals carrying homozygote mutant alleles had the heaviest body weight and the highest DLWG. Consequently, these SNP may be useful indicators in the selection of lambs for higher growth rate and meat production

The Long Intron 1 of Growth Hormone Gene from Reeves' Turtle (Chinemys reevesii) Correlates with Negatively Regulated GH Expression in Four Cell Lines.

Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves’ turtle (Chinemys reevesii) have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH) cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp), comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS) of the turtle’s GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO) cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH) gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves’ turtle might correlate with downregulated gene expression.

Detection of Transgene in Tilapia (Oreochromis niloticus) by Polymerase Chain Reaction (PCR) Technique

Polymerase Chain Reaction (PCR) technique using specific primer can be used to detect transgenes. The present study was undertaken to detect salmon growth hormone (GH) gene in transgenic tilapia (Oreochromis niloticus) by PCR. DNA was extracted from F1 Tilapia generated by crossing transgenic parents. Two primers were designed to amplify a part of the region of GH gene sequence, which was used to make transgenic tilapia. To confirm the specificity of the selected primer, PCR was performed on diluted DNAs, extracted from tilapia fin tissues. GH transgene sequences (1500 bp) were successfully amplified from transgenic fish in this study. The specificity of the primers was found to be high in detecting the salmon GH transgenes. The PCR-based method therefore, could be used for fast and easy screening of transgenic fish for this gene.J. Environ. Sci. &amp; Natural Resources, 8(2): 47-49 2015

Comparison of phylogenetic analysis in the natural salmonids by using growth hormone (GH) gene

To days, The Growth hormone (GH) gene is more important in the regulator of metabolism, osmoregulation, reproduction and skeletal growth in Livestock. GH almost in all of animals has been same function that mentioned. This hormone also is exciting skeletal cellular for more growth and replication. In Salmonids for specially, furthermore, GH gene in population of salmonids can be used as the studies of phylogenetics and finding ancient and pedigree of salmonids that some researchers used from GH gene in salmo salar and salmo trutta for studies of phylogenetics. In this study we had done sequence of fragments of GH gene in salmo trutta caspius full length of almost 2048 bp. and deposited in GeneBank (accession number, JN24163) For sequencing of GH gene in the Salmo trutta caspius, first was extracted DNA genomics from bloods and the muscles of salmons, in related to, we designed three pairs of primers from first to end of the GH gene in same sequences from salmo salar and salmo trutta that reported in GeneBank. After sequencing of fragments we analyzed fragments and compared with other sequences in salmonid fishes. In this research our aims, study of amount variation in the between salmo trutta caspius species with Atlantic salmon and also, study amount of phylogenetic variation between Salmo trutta caspius with other salmons regarding to the GH gene.

The Detection of Superior PO Cattle (Ongole Cattle Descendant) Based on The Growth Hormone Gene Sequence

The aim of this research is to detect the PO cattle as superior beef cattle based on conserved sequence of growth hormone gene. DNA of three superior PO bull from BBIB Singosari and ten PO cattles from UPA Pasuruan has been analyzed.. DNA isolation has been conducted by salting out technique, and continued with PCR & sequencing. Based on clustalW allignment using Mega 5.2 software it is seen that among 214bp, out of 329bp read, 58.4% of sequences are same for the 13 cattles; so during almost one hundred years, since Ongole Cattle introduced to Indonesia from Madras (India), there are still 58.4% of the sequences are not changed yet. The conserved sequences are distributed along the growth hormone gene sequence read. The longest conserved sequence is nineteen bp, that is GGGCCTGGGGCGGCCTTCT. The nineteen conserved sequence and others can be used as indicators of superior PO cattle.

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole ( Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole ( Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males ( P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.

Use of Growth Hormone Gene Polymorphism in Selecting &lt;i&gt;Osmanabadi&lt;/i&gt; and &lt;i&gt;Sangamneri&lt;/i&gt; Goats

Goat meat holds tremendous demand irrespective of cultural and religious barriers. Therefore selection of goats for improved growth is advantageous. Growth hormone (GH) is a polypeptide hormone which is the major regulator of growth of animals, encoded by growth hormone gene. Polymorphic GH gene confers more active variants of the hormone enhancing growth. The objectives of the present study were to identify single nucleotide polymorphisms (SNPs) of GH gene and their association with growth traits of goats. DNA samples of 240 goats from two Indian breeds, namely Osmanabadi and Sangamneri were subjected to single strand confirmation polymorphism (SSCP) followed by DNA sequencing techniques with the objectives of studing single nucleotide polymorphisms (SNPs) in GH gene and their association with growth traits. Twenty three and eighteen nucleotide changes were observed in Osmanabadi and Sangamneri goats, respectively in comparison to goat sequence of accession No. 00476. Of them, twelve were seen unique to both goat breeds indicating considerable deviation from exotic goat sequence. The study revealed point mutations of G200T, A815G, A1753, C1763T and A1789G in GH gene sequence of both goat breeds. In contrast, A497G, A499G C500G C501-2 C730T C781T and C2055T were observed specific to Osmanabadi and Sangamneri breeds respectively indicating the possibility of using them as breed specific markers. G200T resulted GG, GT and TT genotypes and of them, GT was associated with heavy body weight and GG with low body weight (P&le;0.05) in both the breeds. Sangamneri GT genotypes were 6.5% taller (P&le;0.05) than GG. Similarly, AG of A815G revealed low body weight. AA of A1753 insertion revealed heavy body weight in both the breeds and 7% longer body in Sangamneri breed. Heterozygote counterparts for C1763T and A1780G SNPs exhibited heavy body weights ((P&le;0.05). It was concluded that SNPs and their association with body weight may be useful in selecting goats for higher growth traits. DOI: http://dx.doi.org/10.4038/tar.v21i4.3316 <em>TAR </em>2010; 21(3): 398-411

Mammalian genome projects reveal new growth hormone (GH) sequences: Characterization of the GH-encoding genes of armadillo ( Dasypus novemcinctus), hedgehog ( Erinaceus europaeus), bat ( Myotis lucifugus), hyrax ( Procavia capensis), shrew ( Sorex araneus), ground squirrel ( Spermophilus tridecemlineatus), elephant ( Loxodonta africana), cat ( Felis catus) and opossum ( Monodelphis domestica)

Mammalian growth hormone (GH) sequences have been shown previously to display episodic evolution: the sequence is generally strongly conserved but on at least two occasions during mammalian evolution (on lineages leading to higher primates and ruminants) bursts of rapid evolution occurred. However, the number of mammalian orders studied previously has been relatively limited, and the availability of sequence data via mammalian genome projects provides the potential for extending the range of GH gene sequences examined. Complete or nearly complete GH gene sequences for six mammalian species for which no data were previously available have been extracted from the genome databases— Dasypus novemcinctus (nine-banded armadillo), Erinaceus europaeus (western European hedgehog), Myotis lucifugus (little brown bat), Procavia capensis (cape rock hyrax), Sorex araneus (European shrew), Spermophilus tridecemlineatus (13-lined ground squirrel). In addition incomplete data for several other species have been extended. Examination of the data in detail and comparison with previously available sequences has allowed assessment of the reliability of deduced sequences. Several of the new sequences differ substantially from the consensus sequence previously determined for eutherian GHs, indicating greater variability than previously recognised, and confirming the episodic pattern of evolution. The episodic pattern is not seen for signal sequences, 5′ upstream sequence or synonymous substitutions—it is specific to the mature protein sequence, suggesting that it relates to the hormonal function. The substitutions accumulated during the course of GH evolution have occurred mainly on the side of the hormone facing away from the receptor, in a non-random fashion, and it is suggested that this may reflect interaction of the receptor–bound hormone with other proteins or small ligands.

Characterization of the first growth hormone gene sequence for a passerine bird—the pied flycatcher (Ficedula hypoleuca)

While the growth hormone (GH) gene has been characterized in a broad range of vertebrates, surprisingly little is known about this gene in birds. In order to extend knowledge of the GH gene in avian species and non-domestic species, the pied flycatcher (Ficedula hypoleuca) GH gene has been sequenced in this study. The overall average pairwise sequence divergence level was 0.08 among all available avian sequences and 0.27 among other taxa. However, the overall genetic organization of the gene is quite conserved. The similarity of the GH gene sequence of pied flycatchers with those of chicken and duck suggests that the rapid bursts of molecular evolution observed in mammalian and fish GH have not occurred during the divergence of passerine and non-passerine birds.

Varying signals of the effects of natural selection during teleost growth hormone gene evolution

The growth hormone (GH) gene of teleost fish exhibits a higher degree of variability compared with other vertebrate groups. However, the different selective constraints at the sequence level are not well understood. In this study, maximum-likelihood (ML) models of codon substitutions were used to investigate Darwinian adaptive evolution of the GH gene in teleost fishes. Complete GH gene sequences of 54 fish species were classified into 4 orders, and the variable nature of GH was examined by determining the dN and dS rate variation and the rates of molecular evolution for each teleost order. The results indicate that although the overall evolution rate for teleost GH is high ((1.15 +/- 0.01) x 10(-9) substitutions/(aa site x y)) compared with the "slow phases" in mammals ((0.21 to 0.28 +/- 0.05) x 10(-9)), the vital structure of this gene has been retained. While the majority of the amino acid changes appear to be due to relaxation of purifying selection, some positively selected sites were detected in regions with no specifically identified role in protein function. The positively selected regions observed in salmoniformes lineage suggests a possible role for positive selection driving functional divergence in paralogous forms of the GH gene after whole-genome duplication in this lineage.

Polymorphism of the growth hormone gene of red deer ( Cervus elaphus)

In mammals, pituitary growth hormone (GH) is usually encoded by a single gene, but in some caprine ruminants there are two GH genes, and higher primates have a cluster of at least 5 GH-like genes. We have previously shown that in several artiodactyls (chevrotain, giraffe, and hippopotamus) there are two GH gene sequences, differing by 5–21 nucleotides (nt), but whether these arise from two distinct gene loci is unclear. We report here that in the red deer ( Cervus elaphus) also there are two main GH gene sequences (designated A and B) differing at about 23 nt. Investigation of DNA from a number of individual animals demonstrated that this variation was due to allelic polymorphism, with individuals carrying either the A-type or the B-type sequence, or both. A- and B-type sequences showed some variation between individuals. The overall difference between the A and B sequences is substantial—greater than that between the GH gene sequences of three distinct bovine species, Bos taurus (ox), Bos indicus (zebu) and Bos grunniens (yak). The biological significance of the presence of two markedly differing GH gene sequences in red deer is not clear, but it is notable that several of the differences between the A and B sequences occur in the 5′ upstream region, which may be associated with differences in gene expression.

Ectopic expression of the CRF-binding protein: minor impact on HPA axis regulation but induction of sexually dimorphic weight gain.

Corticotrophin-releasing factor (CRF) and urocortin possess a high-affinity binding protein. Although the CRF binding protein (BP) can sequester these ligands and inhibit their activity, the endogenous activity of this protein is not understood. Therefore, transgenic mouse lines that over-express the CRF-BP were created. The transgene was constructed by ligating rat CRF-BP cDNA (1.1 kb) between a mouse metallothionein-I promoter (1.8 kb) and a nonfunctional human growth hormone gene sequence (2.1 kb) in a modified pBR322 plasmid and microinjecting the transgene into C57BL/6 x SJL hybrid ova. The transgene was expressed in 50% in both male and female progeny. All transgenic lines were maintained by crossing transgenic animals with wild-type C57BL/6 mates. Reverse-transcriptase (RT) PCR of the CRF-BP transgene showed that it is widely expressed not only in the brain and pituitary, but also peripheral tissues including the liver, kidney and spleen. Transgenic animals of both sexes showed significant increases in weight gain as established by analysis of variance; however, the weight gain profiles for each sex were distinct. High levels of circulating CRF-BP were detected in the transgenic animals, but the basal ACTH and corticosterone levels were not significantly decreased compared to wild-type littermates. The hypothalamopituitary-adrenal (HPA) axis was stimulated by systemic inflammation induced with lipopolysaccharide (LPS). An expected increase in transgene expression was observed and was accompanied by a significant attenuation of ACTH secretion at 3 h after LPS injection in the transgenic males but not the females. These data suggest that HPA axis regulation is significantly affected only with very high circulating levels of CRF-BP. Moreover, this work supports previous studies that implicate CRF and urocortin in the regulation of appetite and the binding protein expression may play a sexually dimorphic role in regulating this and other responses.

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.

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: a quantitative indicator of metal toxicity.

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of 31 metals, 17 were competent for inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD50 in mice. Apparently the positive/negative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.

Microinjection and expression of a mouse metallothionein human growth hormone fusion gene in fertilized salmonid eggs.

Using a microinjection method (Rokkones et al. 1985) deoxyribonucleic acid was introduced into fertilized salmonid eggs. The survival rate after a 28 day period was 91% for injected eggs in comparison to non-injected controls. A gene construct containing the mouse metallothionein promoter fused to the human growth hormone structural gene was microinjected either as a supercoiled plasmid or as a linear sequence. In Southern blot analysis of both 5 and 73 day old dissected rainbow trout embryos, as well as in 1 year old Atlantic salmon, the mouse metallothionein human growth hormone gene sequence was detected together with the chromosomal DNA when micro-injected as plasmid or as linear DNA. After digestion with Bam HI restriction endonuclease, the human growth hormone gene was excised from the high molecular weight DNA fraction. Transcription into human growth hormone specific RNA, as well as translation and release of human growth hormone immunoreactive protein, could be demonstrated in early embryonic stages.