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Expression of the GH receptor (GHR) gene and its binding with GH is essential for growth and fat metabolism. A GT microsatellite exists in the promoter of bovine GHR segregating short (11 bp) and long (16 to 20 bp) allele sequences. To detect SNP and complete an association study of genotype to phenotype, we resequenced a 1,195-bp fragment of DNA including the GT microsatellite and exon 1A. Resequencing was completed in 48 familialy unrelated Holstein, Jersey, Brown Swiss, Simmental, Angus, Brahman, and Brangus cattle. Nine SNP were identified. Phylogeny analyses revealed minor distance (i.e., Key words: bovine, Brangus, deoxyribonucleic acid, growth hormone receptor, single nucleotide polymorphism

Sequence variations in the bovine growth hormone (GH) gene were investigated by single strand conformation polymorphism (SSCP) analysis of seven amplified fragments covering almost the entire gene (2.7 kb). SSCPs were detected in four of these fragments and a total of six polymorphisms were found in a sample of 128 Holstein bulls. Two polymorphisms, a T[right arrow]C transition in the third intron (designated GH4.1) and an A[right arrow]C transversion in the fifth exon (designated GH6.2), were shown to be associated with milk production traits. [GH4.1.sup.c]/[GH4.1.sup.c] bulls had higher milk yield than [GH4.1.sup.c]/[GH4.1.sup.t] (P [less than or equal to] 0.005) and [GH4.1.sup.t]/[GH4.1.sup.t] (P [less than or equal to] 0.0022) bulls. [GH4.1.sup.c]/[GH4.1.sup.c] bulls had higher kg fat (P [less than or equal to] 0.0076) and protein (P [less than or equal to] 0.0018) than [GH4.1.sup.c]/[GH4.1.sup.t] bulls. Similar effects on milk production traits with the GH6.2 polymorphism were observed with the [GH6.2.sup.a] allele being the favorable allele. The average effects of the gene substitution for GH4.1 and GH6.2 are similar, with [+ or -]300 kg for milk yield, [+ or -]8 kg for fat content and [+ or -]7 kg for protein content per lactation. The positive association of [GH4.1.sup.c] and [GH6.2.sup.a] with milk production traits may be useful for improving milk performance in dairy cattle.

Fatty acid profiles and cholesterol content of whole-carcass ground tissue were compared from 26 transgenic (T) pigs expressing a bovine growth hormone gene (bGH) to 26 sibling control (C) pigs. All pigs were fed a common diet and were slaughtered at five different live weights: 14, 28, 48, 68, and 92 kg. The left side of each intact carcass was ground and tissue samples were analyzed for lipid composition and cholesterol content. At 14-kg body weight, carcasses from bGH-T pigs contained 38% less fat, 44% less saturated fatty acids (SFA), 48% less monounsaturated fatty acids (MUFA), and 38% less polyunsaturated fatty acids (PUFA) than C pigs. At 28 kg, bGH-T pigs had 38% less total carcass fat, 42% less SFA, 46% less MUFA, and 24% less PUFA than C pigs. At 48-kg body weight, bGH-T pigs contained 48% less carcass fat, 55% less SFA, 59% less MUFA, and 22% less PUFA than C pigs. At 68 kg, bGH-T pigs had 78% less carcass fat, 78% less SFA, 79% less MUFA, and 53% less PUFA than C pigs. At 92 kg, carcasses from bGH-T pigs contained 85% less carcass fat, 85% less SFA, 91% less MUFA, and 66% less PUFA than those from C pigs. Cholesterol content was not different between bGH-T pigs and C pigs at any of the carcass weights. The trend was for cholesterol content to decrease from the 14- to 92-kg weight group. These results suggest a dilution effect of carcass fat and fatty acids in carcass tissue from bGH-T pigs with increasing live weight.

Continuous intravenous infusion of bovine growth hormone-releasing factor (bGRF) increases milk synthesis in dairy cattle by as much as 46%. We have begun to develop a system for delivery and expression of a synthetic bGRF gene in cultured bovine cells using the provirus of the bovine leukemia virus (BLV). The gene encoding synthetic bGRF, constructed from eight overlapping oligonucleotides, was fused to the whey acidic protein promoter (WAP) or the mouse mammary tumor virus promoter (MMTV). These plasmids, termed pWAP.GRF and pMMTV.GRF, were able to induce transcription of bGRF upon transfection into Madin-Darby bovine kidney (MDBK) cells and induction with a lactogenic hormonal milieu (prolactin, hydrocortisone, triio-dothyronine, insulin) or dexamethasone. When these constructs were cloned into a BLV vector in place of its oncogenic region, and transfected into MDBK cells, bGRF was expressed. Virus particles were prepared from these cultures and used to deliver the bGRF gene by viral infection into fresh MDBK cells. Northern blot analysis of MDBK total RNA revealed a fivefold higher level of expression of bGRF mRNA in transfected cultures than in virally infected cells, and no expression was detected in control cultures. The bGRF peptide was detected in both cell extracts and media samples from transfected cultures but was not detected in cell extracts or media samples from virally infected cells. This provirus construct may prove useful as a delivery system for peptides into cattle. Introduction Continuous delivery of exogenous peptides into food-producing animals to alter their carcass composition or milk production is difficult. Peptides are usually injected either intravenously, subcutaneously, or intramuscularly to prevent the hydrolysis that would normally occur by the oral route of delivery. Response of the animal to a peptide may be inefficient because of the short half-life of the peptide after a single injection. If the gene for a particular peptide could be stably inserted into an animal under the appropriate regulatory control, then it would be possible to deliver the peptide on demand and to bypass the requirement for an invasive delivery system. We have selected the bovine growth hormone-releasing factor (bGRF) gene as a model system to study delivery of peptides because this peptide is galactopoietic (Dahl et al., 1990). It has been necessary to infuse bGRF peptide intravenously for a maximal response in growth hormone secretion to occur (Moseley et al. 1985). Up to a 46% increase in milk yield has been demonstrated upon infusion of bGRF into dairy cows (Dahl et al., 1990); therefore, this peptide is useful for these experiments because it has a readily detected response and a large metabolic effect. We have begun to investigate alternate methods of delivery for bGRF by using the bovine leukemia virus (BLV) retroviral vector. Retroviral vectors have been used successfully for delivery and expression of foreign genes into both mice (Jaenisch and Croker, 1975; Jahner and Jaenisch, 1980; Rubenstein et al., 1986) and larger domestic animals (Squire et al., 1989). Therefore, the objective of our research was to use a retroviral vector as a method of delivery of a synthetic bGRF gene into bovine cells in tissue culture and to examine expression of this gene under the control of two hormonally responsive promoters. Experimental Procedures Tissue Culture Cells. Madin-Darby bovine kidney (MDBK) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were propagated in Eagle minimal essential medium (EMEM) with Earle salts (GIBCO Laboratories, Santa Clara, CA) and 10% horse serum with 1x penicillin/streptomycin solution (5,000 units penicillin/mL, 5 mg of streptomycin/mL in .9% NaCl; Sigma Chemical, St. Louis, MO). Cultures were maintained in 100-mm cell culture dishes at 37|degrees~C with an atmosphere of 5% C|O.sub.2~. Bovine Growth Hormone-Releasing Factor Gene Construction. The nucleotide sequence used for construction of the synthetic bGRF gene was derived from the published amino acid sequence of bGRF (Esch et al., 1983). Eight overlapping oligonucleotides (oligos) were synthesized for this construct. The sequence of each oligonucleotide was determined by Maxam-Gilbert sequence analysis (Maniatis et al., 1982). Each oligonucleotide was annealed to its complementary strand partner and purified on a 1.6% agarose gel to obtain four cassettes. The first two cassettes (containing oligos I and III and II and IV) were ligated using 1 unit |T.sub.4~-DNA ligase at 16|degrees~C overnight in ligation buffer (50 mM Tris |pH 7.4~, 10 mM Mg|Cl.sub.2~, 10 mM dithiothreitol |DTT~, .1 mg/mL of BSA, fraction V) and the resulting product purified by agarose gel electrophoresis. The next two cassettes (containing oligos V and VI and VII and VIII) were ligated and purified as above. Finally, the two combined cassettes were ligated, purified on a 1.6% agarose gel, and then ligated into the EcoRI site of the plasmid pUC19. The resulting plasmid was called pbGRF. Because of the design of the bGRF fragment, ligation into the vector eliminated the EcoRI site at the |Mathematical Expression Omitted~ end of the gene but maintained the |Mathematical Expression Omitted~ EcoRI site for use in further subcloning. The correct sequence of this construct was verified by DNA sequence analysis using Sequenase (United States Biochemical, Cleveland, OH). Selection of Promoters and Subcloning Strategy. The whey acidic protein (WAP) promoter (Campbell et al., 1984) and the mouse mammary tumor virus (MMTV) promoter (Majors and Varmus, 1983) were obtained from the ATCC (ATCC #63005 and 45007). Both promoters increase transcription in response to the addition of prolactin in tissue culture (Campbell et al., 1984; Munoz and Bolander, 1989). In addition, the MMTV promoter increases transcription in response to the addition of dexamethasone (Buetti and Diggelmann, 1981). The .5-kb (kilobase pair) WAP promoter was excised from its original vector with XhoI and KpnI restriction enzymes (Boehringer Mannheim, Indianapolis, IN) and ligated into the KpnI site of pbGRF overnight at 16|degrees~C with 1 unit of |T.sub.4~-DNA ligase and 2 mM ATP in the same ligation buffer described above. The resulting DNA fragment was made circular by filling in the remaining restriction half-sites (XhoI, KpnI) with 2 units of Klenow enzyme and 2 mM of each of the four deoxynucleotides for 15 min at 37|degrees~C, precipitated in ethanol, resuspended in ligation buffer, and ligated at 22|degrees~C overnight with 1 unit of |T.sub.4~-DNA ligase and 2 mM ATP to generate the construct pWAP.GRF. The 1.3-kb MMTV promoter was excised from its original vector with BamHI and subcloned into the BamHI site of pbGRF. Both plasmid constructs were transformed into E. coli strain DH5-alpha. The correct orientation of each promoter relative to bGRF was determined by DNA sequence analysis using Sequenase. Bovine Leukemia Virus Subclones. Promoter-bGRF constructs were subcloned into the bovine leukemia virus (BLV) vector pBLV-913 (Derse and Martarano, 1990). The WAP.GRF fragment was excised from pWAP.GRF with BamHI and EcoRI. The MMTV.GRF fragment was excised from pMMTV.GRF with an EcoRI and a partial BamHI digest because of the presence of additional BamHI sites (Majors and Varmus, 1983). Both fragments were purified by agarose gel electrophoresis (Maniatis et al., 1982). The pBLV-913 was fully digested at the unique EcoRl site at position 7925 in the |Mathematical Expression Omitted~ end of the viral genome then partially digested with BamHI (Sagata et al., 1985). Viral DNA digested at the BamHI site at 7203 in the viral genome was 10,678 base pairs (bp) in length and was isolated by agarose gel electrophoresis. Fragments were cut from the gel with a razor blade, squeezed through a 1-mL syringe, and extracted with phenol to obtain purified DNA. This viral DNA was missing the tax/rex portion of the viral genome, and therefore, could not replicate and was non-oncogenic (Derse and Martarano, 1990). The promoter-GRF fragments were recombined with the tax/rex deletion BLV subclones by ligating overnight as described in the previous section and transformed into E. coli strain DH5-alpha. Resulting clones (pBLV.WAP.GRF and pBLV.MMTV.GRF) were characterized in detail by restriction enzyme mapping. Transfection of pMMTV.GRF and pWAP.GRF Subclones. Transfection of MDBK cells was performed by the calcium-phosphate precipitation method (Stuart et al., 1985). Ten micrograms of each plasmid or no plasmid was transfected into 80% confluent MDBK cells. Calcium-phosphate precipitation proceeded for 12 h, and the medium was removed and fresh medium added. At 36 h after transfection, 1 |mu~g/mL of dexamethasone was added to one of the two plates containing pMMTV.GRF, and a lactogenic hormone milieu (15 |mu~g/mL of prolactin, 1 |mu~g/mL of hydrocortisone, 40 ng/mL of 3,|Mathematical Expression Omitted~,|Mathematical Expression Omitted~-triiodo-L-thyronine, and 400 ng/mL of insulin) was added to one of two plates containing pWAP.GRF. At 48 h after transfection, total RNA was prepared. Control transfections were prepared in identical fashion except no DNA was added to cells. Transfection/Infection with Bovine Leukemia Virus Subclones. The BLV-GRF constructs were cotransfected into MDBK cells by the calcium-phosphate procedure using a helper plasmid, pBLPX-RSPA (Derse and Martarano, 1990), which contains the tax/rex portion of the BLV genome under the control of the Rous sarcoma virus promoter. One hundred micrograms of each BLV-GRF plasmid was transfected into 80% confluent MDBK cells, along with 50 |mu~g of the helper plasmid. Transfection proceeded for 12 h, then the medium was replaced with fresh medium. At 48 h after transfection, cells were stimulated with either dexamethasone or lactogenic hormonal milieu as described above. All media used for stimulation were lacking horse serum (which would rapidly degrade bGRF peptide) but did contain the protease inhibitor |alpha.sub.2~-macroglobulin to minimize the effects of proteolytic enzymes that remained from residual serum. At 60 h after transfection, the growth medium was collected from each plate and viral particles were purified by sterile filtration through a .22-|mu~m syringe filter (Gelman Sciences, Ann Arbor, MI). Two milliliters of the filtrate was frozen for protein analyses (described later) and 8 mL was used to infect fresh MDBK cells. Total RNA was prepared from the transfected MDBK cells as described below. The MDBK cells were incubated in the presence of growth medium filtrate to allow infection (16 h), then new medium was added. At 72 h after infection, media were changed to include either dexamethasone or lactogenic hormonal milieu as described above. At 84 h after infection, growth media were frozen for protein analysis. Total RNA was also prepared from the infected MDBK cells as described below. Preparation and Analysis of RNA. Preparation of RNA was by the proteinase K method as previously described (Stuart et al., 1985). The RNA (15 |mu~g per lane as quantified spectrophotometrically) was subjected to electrophoresis for 4 h at 50 V on 1.5% agarose, 2.2 M formaldehyde gels in 1x MOPS buffer (Stuart et al., 1985). Gels were stained by placing them in .1 M ammonium acetate with 5 |mu~g/mL of ethidium bromide for 30 min. The RNA sizes were determined by comparison to a .24- to 9.5-kb RNA ladder (Bethesda Research Labs, Bethesda, MD). Transfer to Hybond N solid support (Amersham, Arlington Heights, IL) was accomplished by soaking the gel for 45 min in 50 mM NaOH, 10 mM NaCl, neutralizing the gel by soaking in .1 M Tris-HCl (pH 7.5) for 45 min, and then Northern blotting as described (Maniatis et al., 1982). Transfer was monitored by viewing the Hybond N under ultraviolet light and by restaining the agarose gel. In all cases complete transfer was achieved. The RNA was cross-linked to the solid support by exposure to ultraviolet light for 2 min on a transilluminator (Spectroline Model TR-302, 302 nm ultraviolet). The BamHI/EcoRI bGRF fragment isolated from pbGRF was labeled to high specific activity using a commercial random-primed labeling kit (Boehringer Mannheim), and 32P-CTP (3,000 Ci/mmol) (New England Nuclear, Wilmington, DE). This probe was added to a nonaqueous solution and hybridized at 42|degrees~C for 20 h (Stuart et al., 1985). After hybridization, filters were washed for 1 h in 2x SSC (.15 M NaCl, .015 M sodium citrate), .1% SDS (sodium dodecyl sulfate) at 65|degrees~C, followed by 30 min in .3x SSC, .1% SDS at 65|degrees~C. Filters were exposed to x-ray film (X-OMAT, AR, Eastman Kodak, Rochester, NY) at -70|degrees~C with intensifying screens. X-ray films were analyzed with a densitometer (Model 710 Fluorometer/Densitometer, Ciba-Corning, Oberlin, OH). Bovine Growth Hormone-Releasing Factor Immunoblot Analysis. Immunoblots of bGRF peptide were prepared by electrophoresis of concentrated cell extracts and growth media prepared from transfected/infected MDBK cells using SDS-PAGE by the Hoefer procedure for low-molecular-weight protein gels (Giulian and Graham, 1990) with the following modifications: 50 mM DTT was added to the sample buffer, and samples were boiled for 5 min before they were loaded onto the gels. Two gels were used to resolve identical sets of protein samples. One gel was stained overnight in Coomassie brilliant blue G in 50% methanol, 10% acetic acid, then destained in 10% methanol, 10% acetic acid for viewing of samples and molecular weight markers for peptides (2,500 to 17,000) (Sigma). Proteins from the second gel were transferred via capillary action to nitrocellulose. The gel was equilibrated in 50 mM Tris-HCl (pH 9.5) for 10 min and placed on two sheets of Whatman #3 chromatography paper that functioned as wicks for the reservoir buffer (50 mM Tris-HCl 9.5). Zetabind membrane (charged nylon) (CUNO, Meriden, CT) was cut to size, hydrated, and then placed on the gel. Two sheets of Whatman chromatography paper were placed on the membrane and a stack of paper towels placed on the top of them. Passive transfer proceeded for 39 h with a change of transfer buffer and paper towels at 18 h. The primary antibody in the immunoblot analysis was a polyclonal rabbit anti-serum prepared against purified bGRF (1-44)-N|H.sub.2~ (M. B. Kamdar, Upjohn, Kalamazoo, MI). The antibody was characterized by M. B. Kamdar. Cross-reactivity studies were performed by RIA analysis using rabbit anti-bGRF (1-44)-N|H.sub.2~ at an initial titer of 1:40,000 and 125I-hGRF (1-44)-N|H.sub.2~ (Amersham, Lot #53,55) as tracer. The antibody cross-reacted completely with bGRF (1-44)-N|H.sub.2~ and GRF variants with conserved 1-29 ammo acid regions, but cross-reacted only minimally (|is less than~ .01 to 8%) with GRF variants containing |is less than~ 29 amino acids. The secondary antibody was goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase (Sigma # A8025). Nonspecific binding of antibody to the solid support was reduced by blocking with 10% BSA, .5% nonfat dry milk, .05% Na|N.sub.3~ in PBS for 6 h at 45|degrees~C. The blocking solution was decanted, and 5|mu~g/mL of the primary antibody was added in (TBS)-Tween 20 (50 mM Tris |pH 7.5~, .05% Tween 20, .87% NaCl) with 5% fetal calf serum for 2 h at room temperature. The blot was then washed five times with TBS-Tween 20 for 15 min each. The secondary antibody was then added to the TBS-Tween 20 at a 1:2,500 dilution and incubated at room temperature for 2 h. The blot was washed five times in TBS-Tween 20 for 15 min each. The blot was then rinsed for 15 min in AP buffer (.1 M Tris 9.5, .1 M NaCl, 5 mM Mg|Cl.sub.2) and developed in NBT/BCIP reaction mix (33 mg of nitrobluetetrazolium, 16.7 mg of bromochloroindoyl phosphate in 100 mL of AP buffer). The color reaction was stopped by removal of developer and addition of distilled water. Results Expression of Bovine Growth Hormone-Releasing Factor in Madin-Darby Bovine Kidney Cells Transfected with PWAP.GRF and pMMTV.GRF. To analyze the inducibility of the MMTV and WAP promoters in a bovine cell line, we transfected the plasmids pWAP.GRF and pMMTV.GRF into MDBK cells and induced transcription by the addition of dexamethasone or lactogenic hormonal milieu to the tissue culture media. The RNA prepared from these cells was subjected to Northern blot analysis using a radioactively labeled purified bGRF fragment as probe. The presence of bGRF mRNA in the induced cultures showed a clear transcriptional activation of both promoter-bGRF gene constructs. Expression of Bovine Leukemia Virus Subclones. Cotransfection of BLV subclones, pBLV.WAP.GRF and pBLV.MMTV.GRF, into MDBK cells with a helper plasmid that contains the tax/rex genes under control of the Rous sarcoma virus promoter allowed the expression of bGRF from the transfected cultures as well as preparing virus for infection of other cells. Detectable bGRF expression occurred only upon transfection of large amounts of the BLV subclones (100|mu~g of plasmid per 2 x |10.sup.6~ cells) along with a large amount of the helper plasmid (50|mu~g of plasmid per 2 x |10.sup.6~ cells). The bGRF was clearly detectable in the transfected cultures, whereas in the cultures infected with virus prepared from the transfected plates (and minus the helper plasmid), detectable levels of expression were 80% lower for BLV.WAP.GRF and just above background levels for BLV.MMTV.GRF. Expression of bGRF peptide was monitored using immunoblot analysis. The bGRF peptide was produced from the transfected cultures at a detectable level and was found in both cell extracts and in one tissue culture medium, whereas virally infected cultures did not produce enough protein for detection. Discussion We are interested in development of alternative methods for delivery of peptides into animal systems. It is clear that for useful peptide delivery to occur in an animal system, expression of the gene for the peptide must be tied to a regulatory control found in the animal. Otherwise constant expression of peptides could have deleterious effects, as has been noted for other delivery systems (Squire et al., 1989). Our current studies show that the BLV vector can be used to express a small peptide hormone gene under the control of exogenous promoters in a bovine tissue culture cell system. The use of the WAP and MMTV promoters has allowed us to transfect the bGRF gene into MDBK cells and to achieve transient inducible expression by lactogenic hormones. Also, we were able to express these constructs after subcloning them into the BLV provirus and cotransfecting them with the tax/rex helper plasmid. Virus infection produced a much lower level of RNA expression, and no detectable protein expression. Each experiment was reproduced at least once, and the data were essentially similar between studies. After these assays were performed we checked high-molecular-weight DNA prepared from these cultures by a slot-blot analysis and determined that delivery of viral sequences had occurred (data not shown). It is possible that even though viral DNA was delivered to the cells, gene dosage may not have been equivalent between transfected and infected cultures. Inefficient expression may also have happened because of deletions or point mutations that may have occurred during virus production. Furthermore, it is unknown what effect the missing tax/rex gene products may have on transcription of a foreign promoter in the BLV genome, or translation of a foreign gene product. It is also possible that virus infection is inefficient in this cell system, although it is known that the MDBK cell is a high-titer BLV producer after it is transfected with the original provirus (Derse and Martarano, 1990). Future studies should involve testing a number of bovine cell types for efficient delivery of genes and expression, as well as the development of high-titer virus-producing cell lines. Although we believe this system is worth pursuing for delivery of peptides, one of the obvious drawbacks of the BLV system at this time is that it is a complex retrovirus that still requires further study of its genetic components and their function. The usefulness of viral vector delivery systems for delivery of exogenous genes into animal systems is currently being evaluated by a number of investigators. For example, the Moloney murine leukemia virus has been extensively characterized and used successfully for many years to produce transgenic mice (Jaenisch and Croker, 1975; Jahner and Jaenisch, 1980; Rubenstein et al., 1986). However, difficulty in gene delivery and expression of this system into bovine cells in tissue culture (Squire et al., 1989) convinced us to attempt to develop a bovine viral vector for this purpose. Previous work has shown the bovine leukemia virus provirus to be amenable to the construction and use of its genome as a viral vector in cell culture (Derse, 1988; Derse and Martarano, 1990). This virus contains two regulatory proteins, tax and rex, in the |Mathematical Expression Omitted~ portion of its genome (Derse, 1988). The tax protein is essential for viral replication by acting together with cis-acting sequences in the long terminal repeat sequences to regulate initiation of transcription (Derse, 1988; Derse and Martarano, 1990). Unfortunately, tax is known to induce chromosomal damage to the host cell system (Tanaka et al., 1990) and, therefore, cannot be used to construct cell lines that stably express the tax/rex proteins. Development of a high-titer system may, therefore, have to make use of a BLV provirus that contains a selectable marker. The rex protein acts to modulate the amount of structural protein mRNA (Derse, 1988; Derse and Martarano, 1990). Removal of the tax/rex portion of the genome produces a reliable nononcogenic suicide vector. This could prove useful in future studies for delivery of foreign genes into animal cell culture (Derse and Martarano, 1990). Implications Delivery and inducible expression of a small synthetic peptide was obtained upon transfection of the pBLV.MMTV.GRF and pBLV.WAP.GRF plasmids. Virus containing the synthetic bovine growth hormone-releasing factor gene was produced, and this was used to infect bovine cells. The provirus of bovine leukemia virus seems to be an appropriate choice for the future development of a gene delivery system for peptides into bovine cells. Literature Cited Buetti, E., and H. Diggelmann. 1981. Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones. Cell 23:335. Campbell, S. M., J. M. Rosen, L. G. Henninghausen, and A. E. Sippel. 1984. Comparison of the whey acidic protein genes of the rat and mouse. Nucleic Acids Res. 12:8685. Dahl, G. E., L. T. Chapin, S. A. Zinn, W. M. Moseley, T. R. Schwartz, and H. A. Tucker. 1990. Sixty-day infusions of somatotropin-releasing factor stimulate milk production in dairy cows. J. Dairy Sci. 73:2444. Derse, D. 1988. trans-acting regulation of bovine leukemia virus mRNA processing. J. Virol. 62:1115. Derse, D., and L. Martarano. 1990. Construction of a recombinant bovine leukemia virus vector for analysis of infectivity. J. Virol. 64:401. Esch, F., P. Bohlen, N. Ling, P. Brazeau, and R. Guillemin. 1983. Isolation and characterization of the bovine hypothalamic growth hormone releasing factor. Biochem. Biophys. Res. Commun. 117:772. Giulian, G., and J. Graham. 1990. The electrophoretic separation of low molecular weight polypeptides in polyacrylamide gels. In: Electrophoresis Instruments and Accessories, Techniques and Exercises. 1990-1991. p 126. Hoefer Scientific Instruments, San Francisco, CA. Jaenisch, R. H. Fan, and B. Croker. 1975. Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal. Proc. Natl. Acad. Sci. USA 72:4008. Jahner, D., and R. Jaenisch. 1980. Integration of Moloney leukaemia virus into the germline of mice: correlation between site of integration and virus activation. Nature (Lond.) 287:456. Majors, J., and H. E. Varmus. 1983. A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. Proc. Natl. Acad. Sci. USA 80:5866. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning, a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Moseley, W. M., L. F. Krabill, A. R. Friedman, and R. F. Olsen. 1985. Administration of synthetic human pancreatic growth hormone-releasing factor for five days sustains raised serum concentrations of growth hormone in steers. J. Endocrinol. 104:433. Munoz, B., and F. F. Bolander, Jr. 1989. Prolactin regulation of mouse mammary tumor virus (MMTV) expression in normal mouse mammary epithelium. Mol. Cell. Endocrinol. 62:23. Rubenstein, J.L.R., J. F. Nicolas, and F. Jacob. 1986. Introduction of genes into preimplantation mouse germ line using an infectious retrovirus. Proc. Natl. Acad. Sci. USA 83:366. Sagata, N., T. Yasunaga, J. Tsuzuki-Kawamura, K. Ohishi, Y. Ogawa, and Y. Ikawa. 1985. Complete nucleotide sequence of the genome of bovine leukemia virus: Its evolutionary relationship to other retroviruses. Proc. Natl. Acad, Sci, USA 82:677. Squire, K.R.E., J. E. Embretson, and N. L. First. 1989. In vitro testing of a potential retroviral vector for producing transgenic livestock. Am. J. Vet. Res. 50:1423. Stuart, P., M. Ito, C. J. Stewart, and S. E. Conrad. 1985. Induction of cellular thymidine kinase occurs at the mRNA level. Mol. Cell. Biol. 5:1490. Tanaka, A., C. Takahashi, S. Yamaoka, T. Nosaka, M. Maki, and M. Hatanaka. 1990. Oncogenic transformation by the tax gene of human T-cell leukemia virus type I in vitro. Proc. Natl. Acad. Sci. USA 84:1071.

Genetic variations in plasma GH concentrations before and following thyrotropin-releasing hormone (TRH) stimulation and in IGF-I concentrations were studied in 11-mo-old Polish Friesian cattle (104 heifers and 110 bulls). A possible association between stimulated GH release, IGF-I, and the polymorphism in the GH gene causing substitution of leucine-Leu to valine-Val at amino acid position 127 of the protein was also investigated. The GH concentrations were determined in serial plasma samples collected every 15 min from 15 min before to 135 min after intravenous administration of 0.15 [Mu]g TRH/kg live weight. The analysis was performed on three variables: baseline (mean of samples at -15 and 0 min), peak (sample at 15 min after injection) and rate (peak minus sample at 60 min, divided by 45 min). The IGF-I concentrations were measured in plasma samples taken before the TRH stimulation. Additionally, first lactation records from the 75 cows earlier tested for GH release and IGF-I were used to study a possible association of milk production traits with GH genotypes. The data were analyzed by multivariate mixed linear models. The heritability of IGF-I reached a higher value (0.35) than variables baseline, peak, and rate (0.02, 0.14, and 0.14, respectively). The GH variables were positively genetically correlated with each other (0.22 to 0.93), whereas they had negative genetic correlations with IGF-I (-0.26). The Val/Val genotypes reached the highest peak value compared with other GH genotypes (P [is greater than or equal to] 0.01), whereas the Leu / Leu genotypes had the highest IGF-I concentrations (P [is less than or equal to] 0.05). Moreover, the Leu / Val heterozygotes were superior to others in milk and protein yields, whereas the Leu / Leu homozygotes reached the highest fat yield (P [is greater than or equal to] 0.01). We conclude that GH peak, GH rate, and IGF-I are heritable traits in young dairy cattle and are affected by the Leu / Val polymorphism in the GH gene. Key Words: Dairy Cattle, Insulin-Like Growth Factor, Polymorphism, Somatotropin

Polymerase chain reaction (PCR)-restriction fragment length polymorphisms in the bovine growth hormone (GH) gene were analyzed. Oligonucleotides were designed based on the published DNA sequence and used as primers in PCR. Restriction endonuclease MspI digestion of the PCR products, separated on 4% agarose gels, identified two alleles designated C and D. Digestion of the C allele yielded four fragments of 526 bp, 193 bp, 109 bp and 63 bp while the D allele generated three fragments of 635 bp, 193 bp and 63 bp. The lost MspI site was located within intron 3 of the bovine GH gene.