Sequence analysis of the growth hormone gene of the South American catfish Rhamdia quelen.

Rhamdia quelen is an important Neotropical catfish species for fisheries and aquaculture in southern Brazil, where it is called Jandia. Like other native Brazilian species of economic importance, R. quelen genetics needs more attention for animal breeding programs. The growth hormone gene is known to be linked to a number of molecular markers and quantitative trait loci. We sequenced the coding region of the growth hormone gene with the primer walking technique. As in other Siluriformes, the R. quelen growth hormone gene has four introns and five exons, in a 1465-bp coding region. The tertiary structure of the encoded protein was predicted by bioinformatics; it has four α-helix structures connected by loops, which form a compressed complex maintained by two disulfide bridges.

Duplicated growth hormone genes in a passerine bird, the jungle crow (Corvus macrorhynchos).

Molecular cloning, molecular phylogeny, gene structure and expression analyses of growth hormone (GH) were performed in a passerine bird, the jungle crow (Corvus macrorhynchos). Unexpectedly, duplicated GH cDNA and genes were identified and designated as GH1A and GH1B. In silico analyses identified the zebra finch orthologs. Both GH genes encode 217 amino acid residues and consist of five exons and four introns, spanning 5.2 kbp in GH1A and 4.2 kbp in GH1B. Predicted GH proteins of the jungle crow and zebra finch contain four conserved cysteine residues, suggesting duplicated GH genes are functional. Molecular phylogenetic analysis revealed that duplication of GH genes occur after divergence of the passerine lineage from the other avian orders as has been suggested from partial genomic DNA sequences of passerine GH genes. RT-PCR analyses confirmed expression of GH1A and GH1B in the pituitary gland. In addition, GH1A gene is expressed in all the tissues examined. However, expression of GH1B is confined to several brain areas and blood cells. These results indicate that the regulatory mechanisms of duplicated GH genes are different and that duplicated GH genes exert both endocrine and autocrine/paracrine functions.

Growth hormone-related genes from baboon (Papio hamadryas): Characterization, placental expression and evolutionary aspects.

Pregnancy is a complex physiological condition, and the growth hormone (GH)-related hormones produced in the placenta, which emerged during the evolution of primates, are thought to play an important metabolic role in pregnancy that is not yet fully understood. The aim of this study was to identify the genes and transcription products of the GH family in baboon (Papio hamadryas) and to assess these in relation to the evolution of this gene family. GH-related transcripts were amplified using total RNA from placental tissue, by reverse transcription coupled to polymerase chain reaction (RT-PCR). Three different GH-related transcripts were identified in baboon placental tissue, with two encoding chorionic somatomammotropins (CSH) and one the placental variant of GH (GH-2). The CSH transcripts showed some minor allelic variation, and a splice variant of CSH-C that retains its in-frame third intron. Gene sequences for GH-1 (probably representing the GH gene expressed primarily in the pituitary gland), GH-2 and the two CSHs were identified in the baboon genomic database, together with a CSH-related pseudogene. Phylogenetic analysis of the baboon GH-related sequences, together with those of a related Old World monkey, macaque, and ape outgroup (human), showed the equivalence of the genes in baboon and macaque, and revealed evidence for several episodes of rapid adaptive evolution. Many of the substitutions seen during the evolution of these placental proteins have occurred in the receptor-binding sites, especially site 2, contrasting with the strong conservation of the hydrophobic core.

Nibea coibor growth hormone gene: its phylogenetic significance, microsatellite variation and expression analysis.

The growth hormone (GH) gene has been characterized for a number of fishes and used to establish phylogenetic relationships and as a candidate gene for studies of genetic variation in connection with growth traits. In this study, we report the genomic structure of Nibea coibor GH (designated as ncGH) including its 5'-flanking region, being cloned by homology-cloning and chromosome walking methods. The ncGH gene spans approximately 3.0 kb and consists of six exons and five introns, as found for all cloned teleost GH genes with the exception of carps and catfish. The 5'-flanking region contains consensus sequences for a TATA box, a CRE, a pit-1alpha, a TRE, two HNF-3, a ERE and a GRE. Five microsatellites are identified in the ncGH gene and three of them are polymorphic marker. The open reading frame (ORF) of ncGH is 615 bp in length encoding a polypeptide of 204 amino acids with an estimated molecular mass of 23.04 kDa and theoretical isoelectric point of 7.79. The precursor of ncGH consists of a 17 amino-acid signal peptide and a 187 amino-acid mature peptide. The four Cys residues are located at conserved positions (Cys(69), Cys(177), Cys(194) and Cys(202)), and One possible site for N-glycosylation (Asn-X-Ser/Thr motif) is present at Asn(201). The coding region sequence of ncGH is used to align with the sequences of 18 other species from Percoidei and one species from Anabantoidei using Clustal X. A matrix of 612 bp was used to construct the phylogenetic trees using neighbor-joining and maximum parsimony methods. The phylogenetic trees by two methods are identical in most of the clades with high bootstrap support. Every family all forms independent monophyly on the phylogenetic trees, in the family, the different species also forms the monophyly according to the different genera. The results are also identical to those from morphological data, and demonstrated that the GH gene is very suitable for phylogenetic relationship analysis of Percoidei. To validate the predicted exon/intron boundaries, ncGH cDNA is cloned using RT-PCR, and tissue distributions are investigated using semi-quantitative RT-PCR method. The results indicate that the predicted exon/intron is correct, the ncGH mRNA are mainly expressed in pituitary, and weakly expressed in ovary, brain, liver, gill, intestine, muscle and hear, but not expressed in spleen and kidney.

Growth hormone and insulin-like growth factor of naked carp (Gymnocypris przewalskii) in Lake Qinghai: expression in different water environments.

Here, we report the cloning and characterization of growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-II from naked carp (Gymnocypris przewalskii), a native teleost fish of Lake Qinghai in the Qinghai-Tibet Plateau of China. The GH of naked carp encodes for a predicted amino acid sequence showing identities of 63%, 63%, 91% and 94% with cherry salmon, rainbow trout, zebrafish and grass carp, respectively. Compared to common carp and goldfish, evolutionary analysis showed that genome duplication has had less influence on the relaxation of purifying selection in the evolution of naked carp GH. Sequence analysis of naked carp IGF-I (ncIGF-I) and ncIGF-II showed a high degree of homology with known fish IGF-I and IGF-II. To investigate effects of salinity and ionic composition of the aquatic environment on the GH-IGF axis in naked carp, male fish held in river water were assigned randomly to 4 groups: RW (river-water), RW+Na (NaCl in RW), RW+Mg (MgCl(2) in RW) and LW (lake-water) groups. The concentrations of Na(+) in RW+Na and Mg(2+) in RW+Mg were equal to the concentrations of these ions in lake-water. After 2 days of exposure, the plasma IGF-I levels in the RW+Na and LW groups were significantly higher than the control group (RW), and the plasma GH levels of the LW group were also significantly higher than the RW group. The somatostatin (SS) levels in the hypothalamus significantly increased in the RW+Na group. After 5 days of exposure, these hormone levels did not differ significantly among groups. These results indicate that while the plasma GH and IGF-I levels are osmosensitive, the absence of a change in GH secretion in RW+Na might be partly due to a transiently increased release of hypothalamic SS induced by the stress of neutral-saline water. This is the first report of a salinity-induced increase of GH-IGF-I circulating levels in Cypriniformes.

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.

Independent origin of the growth hormone gene family in New World monkeys and Old World monkeys/hominoids.

The growth hormone (GH) gene family represents an erratic and complex evolutionary pattern, involving many evolutionary events, such as multiple gene duplications, positive selection, the birth-and-death process and gene conversions. In the present study, we cloned and sequenced GH-like genes from three species of New World monkeys (NWM). Phylogenetic analysis strongly suggest monophyly for NWM GH-like genes with respect to those of Old World monkeys (OWM) and hominoids, indicating that independent gene duplications have occurred in NWM GH-like genes. There are three main clusters of genes in putatively functional NWM GH-like genes, according to our gene tree. Comparison of the ratios of nonsynonymous and synonymous substitutions revealed that these three clusters of genes evolved under different kinds of selective pressures. Detailed analysis of the evolution of pseudogenes showed that the evolutionary pattern of this gene family in platyrrhines is in agreement with the so-called birth-and-death process.

Effect of temperature on gene expression of the Gh/Igf system during embryonic development in rainbow trout (Oncorhynchus mykiss).

In fish, the GH/IGF system installs very early during development suggesting that this system could promote embryonic growth and development. In contrast to mammals, the embryonic growth rate of poikilotherms depends considerably on the incubation temperature. Therefore, the aim of this study was to determine if variations of embryo growth in response to temperature could be associated with modifications in the gene expression of the GH/IGF system. In this study, using whole mount in situ hybridisation, we demonstrated that embryo incubation temperature (4, 8, and 12 degrees C) did not change the timing of GH-1 and GH-2 mRNA expression in somatotroph cells (stage 24). Similarly, at hatching (stage 30), we did not observe an obvious difference in GH protein and GH-1 and GH-2 transcript amounts in relation to the incubation temperature. Furthermore, from stage 22 to 25, the highest temperature led to a specific up-regulation of IGF-2 (2-fold between 4 and 12 degrees C), and both IGF-RIa and IGFRIb mRNA (1.5-fold between 4 and 12 degrees C), while no difference was observed for IGF-1 mRNA. Conversely, at hatching, the highest temperature specifically down-regulated IGF-2 (3-fold between 4 and 12 degrees C) and both IGF receptor mRNAs (2 fold between 4 and 12 degrees C) present in the head, while no difference was observed in the trunk. Our results demonstrated that different incubation temperatures during trout embryonic development did not change the stage of somatotroph cell appearance. Before hatching, IGF-2 and both IGF receptors, but not IGF-1 mRNA, were specifically up-regulated by high temperatures and could be related to the enhancement of embryonic growth rate.

Genomic structure and sequence of the pufferfish (Fugu rubripes) growth hormone-encoding gene: a comparative analysis of teleost growth hormone genes.

A nested polymerase chain reaction (PCR) technique for amplifying a fragment of the gene (GH) encoding teleost growth hormone has been developed. Using this technique, a fragment of the pufferfish, Fugu rubripes and Arothron maculatus, dwarf gourami, Colisa lalia; guppy, Poecilia reticulata; and goldfish, Carassius auratus GH genes were cloned. The Fugu rubripes (Fugu) gene fragment was used to isolate the GH gene from a Fugu genomic library. The complete nucleotide sequence of a 8.5-kb SacI genomic fragment containing the Fugu GH gene has been determined. The GH gene spans 2.5 kb from the first codon to polyadenylation signal, and contains six exons and five introns similar to the GH genes of salmonids, tilapia, barramundi, flounder and yellowtail. The GH introns contain microsatellite and satellite sequences. The microsatellites found in the fifth intron of the GH gene are also present in the corresponding introns of tilapia, barramundi and flounder GH genes. Southern analysis revealed that the GH gene is a single-copy gene in the Fugu. The promoter region of the Fugu GH gene contains conserved sequences that are likely to be involved in the pituitary-specific expression of the gene. A phylogenetic tree of nucleotide (nt) sequences of all known teleost GH genes has been inferred using the distance matrix method. The topology of this tree reflects the major phylogenetic groupings of teleosts. The intron patterns and repetitive sequences of GH genes can serve as useful natural markers for the classification and phylogenetic studies of teleosts.

Somatostatin receptor subtype gene expression in pituitary adenomas.

Somatostatin (SRIF) exerts its diverse biological effects through a family of membrane receptors. In addition to inhibiting GH secretion, SRIF has antiproliferative effects and has been used clinically in the treatment of pituitary tumors. SRIF receptor (SSTR) expression has recently been identified in pituitary adenomas, and it is unknown whether differential expression of SSTR subtypes predicts clinical responses to SRIF analogs. We therefore determined which SSTR subtype messenger RNAs (mRNAs) are expressed in pituitary adenoma phenotypes and in normal human pituitary tissue using reverse transcriptase-polymerase chain reaction and tested whether expression of specific SSTR subtype mRNA is necessary for SRIF inhibition of GH secretion in human somatotroph adenomas in vitro. Expression of SSTR subtypes 1, 2, and 5 mRNA was identified in all pituitary adenoma types and normal pituitary tissue. In contrast, SSTR3 mRNA was detected in only one somatotroph adenoma as well as in control insulinoma tissue, a tissue known to express SSTR3 mRNA, and was not detected in normal pituitary tissue. SSTR4 mRNA was not detected in any human pituitary tissue. To determine whether specific SSTR subtype mRNA expression is required for SRIF inhibition of GH secretion, five somatotroph adenomas were treated with 10(-7) mol/L SRIF in vitro, and significant inhibition of GH release occurred in all adenomas. All five tumors expressed SSTR2 mRNA and SSTR5 mRNA, and three expressed SSTR1 mRNA. The absence of SSTR1 mRNA expression did not affect the ability of SRIF to suppress GH secretion. We conclude that: 1) human pituitary adenomas and normal pituitary express multiple SSTR gene transcripts; 2) SSTR5 mRNA, which has not been reported in other human endocrine tumor types, is expressed in neoplastic and normal pituitary tissue; and 3) SSTR2 mRNA, SSTR5 mRNA, and variable SSTR1 mRNA are expressed in GH-secreting tumors, which are responsive to SRIF in vitro. Further understanding of SSTR gene expression in pituitary adenomas will facilitate our understanding of the pathogenetic mechanisms of tumorigenesis and may provide a rationale for the use of specific SRIF analogs for clinical application.

A case for drug treatment of obesity.

Several brain receptor systems can modulate food intake. Thus, in a new strategy against obesity, the patient takes submaximal doses of two drugs that act by differing mechanisms.

Cloning of the sole (Solea senegalensis) growth hormone-encoding cDNA.

We report here the complete nucleotide (nt) sequence of a cDNA clone encoding Solea senegalensis growth hormone (sGH) isolated from an expression library prepared from sole pituitary gland poly(A)+RNA. The library was screened using a flounder GH cDNA. The cDNA sequence containing an insert of 769 nt was found to encode a polypeptide of 203 amino acids (aa), including a signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 17 and 119-nt long, respectively. Northern blot hybridization detected a 0.9-kb RNA species. The sGH cDNA sequence shows homologies of 80.9, 76.9, 73.8 and 64.2% with the GH of tuna, gilthead seabream, flounder and rainbow trout.

An overview of the diagnoses in the Kabi Pharmacia International Growth Study.

This paper provides an overview of the diagnoses of patients entered in the Kabi Pharmacia International Growth Study (KIGS). By May 1991, data from a total of 5377 children treated with growth hormone (GH) were included in the main database. Of these children, 2691 were classified as having idiopathic GH deficiency (GHD), 866 as having GHD of known origin, and 1820 as having other causes of short stature. The majority of patients with idiopathic GHD have no history of perinatal trauma. In the patients with GHD of known origin, 137 were congenital cases and 729 acquired GHD. The largest number of congenital cases (114) belonged to the group of central malformations (e.g. septo-optic dysplasia and empty sella syndrome). Of the cases with acquired GHD, 73% were associated with tumours or leukaemia. Other causes of short stature include 12 groups of diagnoses, with more than 150 cases in four of them (idiopathic short stature, 635; defined syndromes with chromosomal aberrations, 337, of which 304 were Turner's syndrome; defined syndromes without chromosomal aberrations, 157; intrauterine growth retardation without stigmata, 366). Analysis of the KIGS data allows modern GH therapy for GHD to be compared with older treatment modalities. The study offers the advantage of larger numbers of cases than can be achieved in individual trials and allows assessment of the use of GH therapy for GHD of comparatively uncommon causes.

Plasma transport of the 20,000-dalton variant of human growth hormone (20K): evidence for a 20K-specific binding site.

The 20,000-dalton variant of human GH (20K) is known to circulate in blood, but few details are known about its plasma transport. A substantial portion of 20K appears to be protein bound despite its low affinity for the receptor-related GH-binding protein (BP). We, therefore, investigated the binding pattern of 20K in human plasma. Radioiodinated 20K was incubated with plasma or plasma fractions, and the mixture was fractionated by gel filtration. Saturation/Scatchard analysis was performed with both 20K and 22,000-dalton GH (22K). The majority (approximately 80%) of protein-bound 20K was complexed with a low affinity BP (Ka = approximately 2 x 10(5) M-1), with the remainder complexed with the high affinity2 receptor-related BP. The binding of 20K to the low affinity BP was specific for 20K; it may involve a 20K-specific binding site on the previously described low affinity plasma BP (peak I) or a separate 20K-specific BP. 22K did not interact with this binding site/BP. Analysis of the 20K-BP complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing suggested, but did not prove, the existence of a separate BP for 20K. We conclude that 20K in plasma binds primarily to a low affinity, 20K-specific binding site (possibly a new, third GH-BP), with only a small fraction bound to the GH receptor-related BP. This binding pattern is markedly different from that of 22K, which is predominantly bound to the receptor-related BP. The existence of a 20K-specific binding site/BP suggests an as yet unrecognized role for this GH variant.

Certain large forms of circulating immunoreactive human growth hormone are in fact immunoglobulins.

To explain frequent discordances between serum GH levels and clinical manifestation of acromegaly, we investigated the possibility that certain immunoglobulins G (IgGs) might be responsible for the displacement of [125I]human (h) GH in the hGH RIA. We incubated dilute sera from seven active acromegalics (basal immunoreactive hGH, 22-313 micrograms/L) with rat adipocyte plasma membranes adsorbed on polystyrene plates. IgGs that bound to GH receptor sites in the absence and presence of 250 nM hGH (for nonspecific binding) were detected using anti-hIgG (Fc-specific) antibody conjugated with alkaline phosphatase. In this system two of the seven sera studied tested positive for IgGs against GH-binding sites (serum 4 in 1:400 dilution, and serum 7 in 1:10 dilution). We studied further the serum with the highest titer. On Sephadex G-100, most of the GH-like immunoreactivity (assayed by RIA) present in serum 4 coeluted with IgGs (assayed by immunodiffusion) as a high mol wt (greater than or equal to 150 kDa) component. To confirm its IgG nature, this material was then adsorbed on protein-A-Sepharose and eluted with 0.1 M sodium citrate, pH 3.0. The protein-A-purified IgGs from serum 4 bound specifically to GH receptor sites in adipocyte membranes and displaced [125I]hGH in the hGH RIA. In contrast, IgGs purified from another acromegalic patient (313 micrograms/L hGH) repeatedly tested negative in the membrane binding assay and hGH RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Three types of growth hormone cells of the rat anterior pituitary as revealed by immunoelectron microscopy using a colloidal gold-antibody method.

Immunoelectron microscopy using a colloidal gold-antibody method with anti-rat GH serum demonstrated three morphologically different types of GH cells in the rat anterior pituitary. They were distinguished as Types I, II and III GH cells, containing only large secretory granules about 350 nm in diameter, mixed large and small granules, and only small granules about 150 nm in diameter, respectively. Double gold labeling with large gold particles for GH and small particles for PRL or ACTH indicated that neither GH and PRL nor GH and ACTH were contained in the same cell. In adult male rats, Type I cells (68%) predominated over Type II (22%), while Type III cells were rare (9.7%). On the contrary, in the adult female rats, Type II cells (47%) slightly dominated over Type I (44%) though the rate of Type III cells was the same as in the male. In neonatal infants, the frequency of occurrence of Type III cells was as high as about 20%; sex differences between Types I and II were indistinct. The Type III cells were therefore thought to represent an immature type.

Molecular forms of circulating growth hormone during spontaneous secretory episodes and in the basal state.

After pharmacological stimulation of the pituitary gland, human GH (hGH) in plasma consists of three or more monomeric molecular forms and several corresponding oligomers. However, the chemical nature of hGH circulating under physiological (stimulated or basal) conditions is not known. In particular, the molecular basis for the GH-like bioactivity of plasma is poorly understood. To gain information on the type(s) of hGH normally found in blood, we extracted hGH from plasma obtained at the time of spontaneous secretory episodes (nocturnal and random release) and during basal periods in 15 normal subjects. Appropriate plasma volumes (30 or 300 ml) were extracted by immunoadsorbent chromatography, and the extracts were analyzed by native polyacrylamide gel electrophoresis at pH 7.5 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 10. The plasma hGH pattern at the time of spontaneous secretion was similar to that after pharmacological stimulation and consisted of 22K (principal), 20K, and acidic hGH as well as hGH dimer. In contrast, plasma hGH patterns during basal periods were highly variable and included immunoreactive hGH fragments in addition to the known hGH forms. Components with mol wt of 30K, 16K, and 12K were consistently identified. We conclude that 1) endogenously stimulated hGH secretion results in the same plasma hormone patterns as pharmacological stimuli; 2) several immunoreactive hGH fragments contribute to the heterogeneity of plasma hGH; and 3) hGH fragments may become a dominant part of total immunoreactivity in the basal state.