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.     

Synthesis of bovine growth hormone by Streptomyces lividans

Streptomyces lividans 66 was transformed with a plasmid containing the regulatory region of the Streptomyces fradiae aph gene and a structural gene that specifies bovine growth hormone (bGH). When grown in liquid culture the transformant contained a protein identical to authentic bGH, as judged by radioimmunoassay and immuno-blotting (Western analysis). The bGH was present in cells that had been in culture for up to four weeks but was not found in the medium. The strategy employed should be generally applicable to the expression of foreign genes in actinomycetes.     

The Expression of CAT Gene in Transgenic Tomato Plant

Bacterial CAT gene .with the 35S promoter of CaMV was transferred into leaf discs of cultivar Lycopersicon esculentum 462 with the help of Ti plasmid of Agrobacterium turnefaciens. These leaf discs were placed on MS salt and B5 vitamine medium containing kanamycin and carbenicillin. Several kinds of phytohormones were chosen in the medium, and it was found that zeatin and NAA have great effects on shooting and rooting. Transgenic tomato plants showed their normal flowering and fruiting. Leaves of these transgenic tomato plants were used for assaying the gene expression. Resuits of Southern Blot showed that the CAT gene was stably integrated into the genome of transgenic plants, The bacterial CAT, proteins were also detected, in transgenic tomato leaves with immunoreaction of CAT antibody. The methods presented here for culturing transformed tomato ceils will be a great help for transfering economically important genes into cultivars of tomato plants in China.     

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.     

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.     

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.     

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.     

干旱耐逆基因（HS1）转化甘薯获得转基因植株

以甘薯（1pomoeabatatas（L.）Lam．）品种栗子香的胚性悬浮细胞为受体材料,用根癌农杆菌介导法,获得了表达除草剂抗性基因bar基因的转HSl基因甘薯植株。共计380个遗传转化的胚性细胞团,在添加2mg／L2．4-D、100mg／L Carb和10mg／L Glu（glufosinate）的固体Ms培养基上选择培养9周后,得到了12个Glu抗性愈伤组织。将这些抗性愈伤组织转移到添加1mg／L ABA、100mg／L羧苄青霉素和10mg／L Glu的固体MS培养基上,其中的3个抗性愈伤组织再生出拟转基因植株。PCR鉴定它们为转基因植株。Southern blot分析表明,HS1基因已整合到基因组中。转基因植株具有稳定的除草剂抗性。结薯观察实验结果表明,转基因植株结薯正常。     

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。     

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.     

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.     

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.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

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.     

Study on the conditions of Cell Transformation in Astragalus sinicus

The conditions of genetic transformation of cells in Astragalus sinicus were studied. The experimental results showed that Agrobacterium tumefaciens strain C58 (pKIW 105), when incubated in medium of low pH and low phosphate concentration in presence of acetosyringone could be induced and activated. When the activated bacteria were used to infect A. sinicus, the GUS gene transient expression in the hypocotyl protoplasts of A. sinicus was immediately and remarkably enhanced. This indicated that the vir gene of A. tumefaciens was activated under the above-mentioned incubation conditions which facilitated T-DNA transfer. In PEG-mediated DNA direct transfer, transient expression of GUS gene was promoted by higher pH and higher Ca2+ concentration of fusion medium. In the same experimental condition, expression of GUS gene under the control of MAS-CaMV 35S chimeric promoter was more effective than that under the control of CaMV 35S promoter, and intensity of GUS gene expression was positively correlated with the amount of foreign plasmid DNA in the range of 10--100 μg. Adventitious shoots were induced from cotyledon and hypocotyls explants treated with Agrobacterium turnefaciens strain PGV 2260 (pBI 121) and were subcultured on MS medium containing 50 mg/L kanamycin to select transformants, and then the transformed shoots were rooted. Stable expression of the foreign genes in the transformed plants was confirmed by assay of neomycin phosphotransferase Ⅱ (NPT Ⅱ ) and β-glucuronidase (GUS) activity.