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.     

榕江香猪生长激素基因的鉴定及功能分析

生长激素是调节动物生长的主要激素.本研究应用聚合酶链式反应技术从榕江香猪的基因组文库中分离出1.903kb生长激素基因.克隆的生长激素基因由五个外显子和四个内含子组成.榕江香猪生长激素基因的碱基序列与已知四个国外猪种和9个中国地方猪种之间的同源性为97%～99%,其间的差异主要集中在内含子2和4.通过限制性内切酶（DdeI,NarI,BsmNI）分析,鉴定出榕江香猪生长激素基因的五个多态性位点,分别位于5＇-侧翼区274（T/C）位点,外显子2的622（G/A）和631（G/A）位点,内含子2中的841（T/C）以及外显子4中的1 358（A/G）位点.同时,1 358（A/G）位的碱基改变导致榕江香猪生长激素成熟肽第108位异亮氨酸替换,三维结构分析表明,异亮氨酸的存在可能导致生长激素与受体间亲合力降低.     

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.     

Rainbow trout has two genes for growth hormone

We report the primary structures of two mRNA species (GH1 and GH2), each predicted from the cloned cDNA and genomic gene sequences, that encode growth hormone in rainbow trout (Salmo gairdneri). Both GH1 and GH2 mRNA contain open reading frames comprising 630 nucleotides and encode 210 amino acid residues, of which 11 are variant. The translated regions of mRNA are flanked by a short 5'-untranslated sequence, which is highly conserved, and a relatively long 3'-untranslated sequence, which is highly divergent. The differences at the 3'-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. RNA blot analysis of trout pituitary RNA using an oligonucleotide probe specific for the GH2 sequence indicates that the cloned gene is expressed. The GH1 and GH2 mRNA likely are transcribed from two distinct loci, which were duplicated during tetraploidization of the salmonid genome between 50 and 100 million years ago.     

Autotransgenic and allotransgenic manipulation of growth traits in fish for aquaculture: a review

It is noteworthy that the use of transgene elements homologous to both the structural gene and promoter region sequences are more effective than heterologous ones for growth hormone (GH) transgenesis in farmed fish species. The generation of autotransgenic fish carrying the GH-transgene construct (of which elements originate from the same species only) has been given considerable attention as a potential means to produce a desirable fish strain acquiring significantly improved growth phenotype. Currently, several growth-enhanced autotransgenic lines including mud loach, carp and tilapia, have been developed or are ongoing for aquacultural purpose. This review considers the generation, development and prospects of autotransgenic manipulation of fish growth, comparing the growth performance of currently available autotransgenic fish strains with those of relevant fast-growing allotransgenic fishes.     

Promoter shuffling has occurred during the evolution of the vertebrate growth hormone gene

Comparative studies of vertebrate gene promoter regions seldom detect gross rearrangements ('promoter shuffling') since such analyses usually employ relatively similar DNA sequences. Conversely, attempts to compare evolutionarily more divergent promoter sequences have been largely unsuccessful owing to the inability of conventional alignment procedures to deal with gross rearrangements. These limitations have been circumvented in the present study by using the novel technique of complexity analysis to identify modular components ('blocks') in the growth hormone (GH) gene promoter sequences of some 22 vertebrate species, from salmon to human. Significant rearrangement of blocks was found to have occurred, indicating that they have evolved as independent units. Some blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Considerable variation between orthologous GH gene promoters was apparent in terms of block length, copy number and relative location. It may be inferred that a wide variety of different mutational mechanisms have operated upon the GH gene promoter over evolutionary time. These include gross changes such as deletion, duplication, amplification, elongation, contraction, transposition, inversion and fusion, as well as the slow, steady accumulation of single base-pair substitutions. Thus the patchwork structure of the modular GH promoter region, and those of its paralogous GH2 and prolactin (PRL) counterparts, have continually been shuffled into new combinations through the rearrangement of pre-existing blocks. Although some of these changes may have had no influence on promoter function, others could have served to alter either the level of gene expression or the responsiveness of the promoter to external stimuli.     

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)     

Inhibition of rat growth hormone promoter activity by site-specific DNA methylation

The effect of methylation on rat growth hormone (rGH) promoter activity was determined in GH3 cells by measuring rGH-Neo and rGH-CAT fusion gene expression with or without prior in vitro treatment with the site-specific DNA methyltransferases, M-BsuE and M-HhaI. To assay for rGH-promoter-specific effects of DNA methylation, RSV-Neo and RSV-CAT activities with or without M-BsuE, M-HhaI and M-HpaII treatment were measured in parallel cultures of GH3 cells. GH1-Neo and RSV-Neo fusion gene expression was inhibited by in vitro methylation from 44 to 83% as measured by the number of Geneticin-resistant GH3 cell colonies. Methylation of the GH1 promoter by M-BsuE exhibited some selective inhibition of Neo expression as determined by colony numbers, although extensive methylation of non-promoter DNA in GH1-Neo and RSV-Neo by M-HhaI and M-HpaII also inhibited Neo expression. Southern blot analysis of genomic DNA isolated from the Geneticin-resistant GH3 cells indicated that Geneticin-resistance was accompanied by demethylation of the BsuE (ThaI) sites in stably incorporated GH1-Neo DNA but not RSV-Neo DNA. Transient expression of the CAT gene in GH3 cells was selectively inhibited by 60% upon methylation of two BsuE (ThaI) sites in the GH1 promoter of GH1-CAT by M-BsuE. The data demonstrate, for the first time, to our knowledge, a direct effect of DNA methylation on the activity of the rat growth hormone promoter.     

Insulin-like growth factor-I (IGF-I) attenuation of growth hormone is enhanced by overexpression of pituitary IGF-I receptors.

Insulin-like growth factor-I (IGF-I) attenuates GH gene expression by a receptor-mediated mechanism in pituitary cells. We, therefore, isolated neomycin-resistant stable GC cell transfectants over-expressing human IGF-I receptor cDNA (IGFIR-cDNA) cloned in an Rous sarcoma virus-directed expression vector. A transfection control contained the IGFIR-cDNA cloned in the reverse orientation. Southern analysis confirmed incorporation of human IGFIR-cDNA sequences into rat genomic DNA. Immunoprecipitation of metabolically labeled [35S]methionine stably transfected cells revealed a 200-kDa human IGF-I receptor precursor protein. Growth rate and basal GH secretion were not altered in transfected cells. Although transfected and control cells had a similar Kd for IGF-I binding (0.43 and 0.40 nM, respectively), IGF-I-binding sites were induced 17-fold (384,000 vs. 22,000 sites/cell). Treatment of cells with IGF-I (6.5 nM) maximally attenuated GH secretion by 80% compared to 40% attenuation in control cells (P less than 0.0001). Maximal suppression of GH in transfectants occurred within 15 h of treatment, and GH secretion by control cells was only maximally suppressed after 42 h. The ED50 of IGF-I suppression of GH secretion in transfectants after 15 h was 0.5 nM. These results demonstrate that transfectants overexpressing human IGF-I receptor are hyperresponsive to exogenous IGF-I. These data indicate that IGF-I receptor number plays an important role in mediating the signal transduction of IGF-I to the GH gene.     

Growth hormone locus expands and diverges after the separation of New and Old World Monkeys

While most mammals including the prosimians have a single copy of the growth hormone (GH) gene, anthropoids possess a cluster of GH-related genes. Throughout the evolution of the main anthropoid groups [New World Monkeys (NWM), Old World Monkeys (OWM), and apes], two features stand out of the GH loci. The first is the appearance of chorionic somatommamotropin hormone (CSH) genes within the OWM lineage and the second is the expansion of the loci intergenic regions in the OWM and apes. In relation with this loci expansion, the NWM possess intergenic regions of homogeneous lengths (3.5 kb). In contrast, heterogeneous lengths (6 and 13 kb) have been reported for species of the OWM. At the present, none of the OWM genomic GH loci organizations have been described. Here, we report the genomic organization of the GH locus in the rhesus monkey, this locus has six GH-related genes separated by five intergenic regions. The 5' end gene (GH-1) encodes for the pituitary GH and is followed by CSH-1, GH-2, CSH-2, CSH-3 and CSH-4 genes. The five intergenic regions have heterogeneous lengths and also present more or less the same Alu distribution as the human GH locus. To analyze the events that contributed to the extension of the intergenic regions of the GH locus and the emergence of the regulatory elements, the five GH locus intergenic regions of the spider monkey (NWM) were sequenced. The results of comparing the loci from both species suggest that the long intergenic regions (13 kb) of the rhesus GH locus share a common ancestor with the 3.5 kb intergenic regions of the spider monkey. However, the observed increased length of the former is due to an insertion (approximately 8.7 kb) at their 3' end. Interestingly in this insert, we discovered a DNA element resembling the enhancer of the CSH genes of the human GH locus. On the other hand, we observed that the short intergenic regions (6 kb) increased by a different recombination event.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

A chromosome‐level genome assembly of the giant grouper (Epinephelus lanceolatus) provides insights into its innate immunity and rapid growth

The giant grouper (Epinephelus lanceolatus) is the largest coral reef teleost, with a native range that spans temperate and tropical waters in the Pacific and the Indian Oceans. It is cultured artificially and used as a breeding species in aquaculture due to its rapid growth rate. Here we report a giant grouper genome assembled at the chromosome scale from sequences generated using Illumina and high‐throughput chromatin conformation capture (Hi‐C) technology. The assembly comprised 1.086 Gb, with 98.4% of the scaffold sequences anchored into 24 chromosomes. The contig and scaffold N50 values were 119.9 kb and 46.2 Mb, respectively. The assembly is of high integrity, including 96.4% universal single‐copy orthologues based on BUSCO analysis. Through chromosome‐scale evolution analysis, we identified alignments of six giant grouper chromosomes to three stickleback chromosomes and some of the genes located within the breakpoints of reshuffling events may related to development and growth. From the 24,718 protein‐coding genes, we found that several gene families related to innate immunity and glycan biosynthesis were significantly expanded in the giant grouper genome compared to other teleost genomes. In addition, we identified several genes related to the hormone signalling pathway and innate immunity that have experienced positive selection or accelerated evolution, implicating their roles in immune defence and fast growth of the species. The high‐quality genome assembly will provide a valuable genomic resource for further biological and evolutionary studies, and useful genomic tools for breeding of the giant grouper.