Synthesis of bovine growth hormone in primates by using a herpesvirus vector.

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.     

Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.     

High-level expression of the bovine growth hormone gene in heterologous mammalian cells

The gene coding for bovine growth hormone (bGH) was isolated from a lambda-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.     

Association of DNA polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and black-and-white cattle

Polymorphisms of the prolactin (bPRL) and growth hormone (bGH) genes were studied comparatively in the Russian and German Black-and-White and Yaroslavl cattle breeds. Two polymorphisms were studied for each gene. In the case of the bPRL gene, the polymorphism of the 5'-untranslated region was examined by microsatellite analysis and the RsaI polymorphism of exon 3, by RFLP analysis. In the case of the bGH gene, the MspI polymorphism of intron III and the AluI polymorphism of exon 5 were assessed by RFLP analysis. Differences in allele and genotype frequencies were observed both between and within breeds. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed; that at the microsatellite of the bPRL gene was low (3.2-24%) in all breeds examined. Homozygotes BB at the bPRL gene, which had not been reported earlier for European cattle breeds, were detected in the German Black-and-White and Yaroslavl breeds (at frequencies 0.16 and 0.13, respectively). The frequency of allele MspI(-) of the bGH gene in the Yaroslavl breed was extremely low (0.02), comparable only with that of the Holstein cattle (0.02). The heterozygosity at the AluI polymorphism was higher than at the MspI polymorphism of the bGH gene and reached 55% in the Yaroslavl breed. Genotype BB of the RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk. The genotypes of the AluI polymorphism of the bGH gene were associated with the fat content in milk in the Yaroslavl (F = 4.56, P = 0.013) and German Black-and-White (F = 4.1, P = 0.014) breeds: the highest fat content in milk was observed in the subsample of cows with heterozygous genotype VL.     

Association between Sscp Haplotypes at the Bovine Growth Hormone Gene and Milk Protein Percentage

The bovine Growth Hormone gene (bGH) is an attractive candidate gene for milk production in cattle. Single-strand conformation polymorphisms at bGH were identified and used to define haplotype configurations at this gene in the Israeli Holstein dairy cattle population (Bos taurus) and in the parent animals of the International Bovine Reference Family Panel (a collection of B. taurus and B. indicus crosses). B. taurus and B. indicus haplotypes at the bGH gene differed qualitatively, confirming the previously proposed long evolutionary separation of these cattle subraces. Only a small number of bGH haplotypes were present in the Israel Holstein population. One of the haplotypes, apparently of B. indicus origin, was found to have a highly significant positive effect on milk protein percentage. This illustrates the utility of the haplotype approach for uncovering candidate gene involvement in quantitative genetic variation in agricultural populations. The strong effect of an indicine haplotype in a taurine background raises the possibility that indicine alleles at other candidate genes may comprise a genetic resource for improvement of taurine populations. It is proposed that haplotype analysis may be a useful adjunct to measures of genetic distance for evaluating rare breeds with respect to gene conservation.     

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Summary. Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' Eco RI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele

Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' EcoRI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.     

Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

用原位分子杂交技术定位牛生长激素基因于5号染色体

本工作利用放射性标记的ｂＧＨ基因（３．０ｋｂ）为探针,通过原位杂交定位牛生长激素基因于染色体５ｑ２２－２６内。该结果与以前的ｂＧＨ基因定位的结果不同,讨论了基因探针、基因定位方法等方面与定位准确性的关系。     

Enhanced expression of bovine growth hormone gene by different culture conditions in Escherichia coli

To optimize the production of bovine growth hormone (bGH) in E. coli, the cells harboring pUBJ10 plasmid, which contains the modified 59-coding region of bGH cDNA under the control of trc promoter, was induced to express under various culture conditions such as medium (LB or M9CA), temperature, induction stage, expression time, IPTG concentration, and hosts. Induction stage was effective at early logarithmic phase. The expression levels of bGH were not largely affected by IPTG concentrations, slightly greater in LB medium than in M9CA medium, and efficient in 4 to 6 h of expression time. The highest level of bGH production was obtained in E. coli BL21 strain.     

The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli

In order to increase the synthesis of bovine growth hormone (bGH) using T7 promoter system in E. coli, the artificial AT-rich block was introduced into the upstream region of a consensus Shine-Dalgarno (SD) sequence and the spacer region (between SD and ATG codon) was enriched with A and T nucleotides. The cells harboring pTAJ plasmids with AT-rich block produced bGH in the range of 3% to 25% and the cells harboring pTBJ plasmids with AT-rich sequence in the spacer region from 0.8% to 20% of total cell proteins. This result suggests that AT rich block and AT nucleotides in the spacer region destabilize mRNA secondary structure, depending on the downstream coding information of bGH gene and also, implying that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.     

Functional expression of bovine growth hormone gene in Pleurotus eryngii

The expression of the bovine growth hormone (bGH) gene was examined in Pleurotus eryngii, which belongs to the family of oyster mushrooms. The region encoding mature bGH, which has a variety of regulatory effects on growth and metabolic processes, was amplified using designed primers containing initiation and termination codons and then subcloned into pPEV binary expression vector. The recombinant vector (pPEVbGH) was introduced in P. eryngii via Agrobacterium tumefaciens-mediated transformation. Recombinant bGH was expressed in P. eryngii harboring pPEVbGH vector under control of the cauliflower mosaic virus (CaMV) 35S promoter up to a level of approximately 26% of total cell proteins after 6 days of cultivation, after which the recombinant protein was analyzed by SDS-PAGE and Western blotting. Interestingly, the growth rate of P. eryngii mycelia harboring pPEVbGH vector was approximately three times faster than that of control P. eryngii, suggesting that bGH affected the growth of P. eryngii. Biological activities were examined in Sprague-Dawley rats, which were administered regular feed mixed with mycelial extracts containing bGH (0.1 or 0.2 μg of bGH per g of animal feed). Mycelial extracts containing bGH significantly affected growth rates and lipid profiles; total cholesterol, triglyceride, HDL, and LDL levels were improved in rats fed mycelial extracts compared with those administered regular feed containing nontransgenic P. eryngii. This result indicates that P. eryngii harboring pPEVbGH vector could produce biologically active bGH. Further, levels of all growth-related factors increased, resulting in faster growth rates in bGH-treated groups. Accordingly, these data suggest that P. eryngii can be applied to the production of industrially useful proteins using a plant expression vector as an efficient mushroom host system.     

Differential in vivo activities of bovine growth hormone analogues

In rodents, bovine (b) growth hormone (GH) binds only to GH receptors, while human (h) GH binds to both GH and PRL receptors. The phenotypic consequences of expression of bGH and hGH in transgenic mice are different and, in some cases, opposite. In the present study, site-directed in vitro mutagenesis of the bGH gene was used systematically to eliminate its differences from hGH at one, two, three or four sites suspected of conferring lactogenic activity: D11, H18, S57 and T60, respectively (corresponding to sites 12, 19, 57 and 60 of the bGH molecule). The resulting bGH analogues were expressed in cell lines and in transgenic mice. All of the seven bGH analogues produced retained their ability to bind to GH receptors and exhibited somatogenic activity in vitro and in vivo. However, none of them were able to bind to PRL receptors or to elicit detectable lactogenic response in vitro. Transgenic animals expressing any of the generated analogues were characterized by gigantism and splanchnomegaly. The effects of expression of each of the double, triple or quadruple mutants on the seminal vesicle weight resembled the effects of wild-type hGH and differed from the effects of expression of wild-type bGH. There were differences between the effects of the expression of different bGH analogues on plasma PRL levels and on the PRL response to pharmacological blockade of catecholamine synthesis. Plasma LH levels in ovariectomized females were suppressed by several of the analogues tested, an effect not seen in animals expressing wild-type bGH or hGH. Dopamine turnover in the median eminence of male mice was also altered in animals expressing different bGH analogues but not in those expressing wild-type bGH or hGH. In ovariectomized females, the effects of different bGH analogs on the turnover of dopamine and norepine phrine in the median eminence included changes resembling those detected in animals expressing hGH, as well as alterations differing from the effects of bot h bGH and hGH.The results indicate that biological actions of these bGH analogues cannot be characterized simply in terms of enhanced or reduced somatogenic or lactogenic activity and raise a possibility that different sites, domains or features of the tri-dimensional structure of GH are involved in its actions on different cellular targets     

Reproductive performance of a transgenic boar carrying the bovine growth hormone gene (bGH)

The aim of this study was to obtain information on the possible influence of the bovine growth hormone gene (bGH) on gametogenesis and reproductive parameters of a 2-year-old Polish Landrace transgenic boar. The bGH gene construct had been introduced into the zygote of the boar with the use of the microinjection technique. On the basis of the available documentation we established that the fertility of the investigated transgenic (bGH) boar was low in comparison with other animals of the Polish Landrace breed, with a poor libido, ineffective matings and, on average, 3 live piglets less per litter. Samples of testis tissue from the boar were obtained after castration. In total, we observed 100 spermatocytes and all of them had normally paired bivalents. It is possible that the boar's lower fertility was caused by some, as yet unknown factor.