Glycine 119 of bovine growth hormone is critical for growth-promoting activity.

Glycine 119 of Bovine Growth Hormone is Critical for GrowthPromoting Activity

Wen Y. Chen, David C. Wight, Bhavin V. Mehta, Thomas E. Wagner, and John J. Kopchick Department of Zoology Molecular and Cellular Biology Program Edison Animal Biotechnology Center and College of Engineering and Technology (B.V.M.) Ohio University Athens, Ohio 45701

drate metabolism (14-20). The multiple physiological effects of GH both in vitro and in vivo have been well documented (13,18, 20,21); however, advances in the understanding of the mechanism of GH action remain elusive. Explanations of the ability of one hormone to direct multiple biological effects have focused on the existence of tissue-specific subtypes of GH receptors (21-22) or the existence of multiple active domains within the GH molecule (23-24). Previously, we have demonstrated that the third ahelix of bGH (residues 109-126) is important for growth enhancement in transgenic mice. Substitution of proline residues, known a-helix breakers, in this region abolished the growth-promoting activity of bGH in transgenic mice despite the presence of high serum levels of bGH analogs (25). We also have demonstrated that three amino acid substitutions in the third a-helix of bGH, bGH-E117L-G119R-A122D(bGH-M8), destroyed the growth-promoting activity of the molecule in transgenic mice. In addition, expression of relatively high levels of bGH-M8 results in dwarf transgenic mice (DTM; 26). Growth suppression in DTM is correlated with serum levels of bGH-M8 and directly related to serum insulin-like growth factor I levels (27). Since bGHM8 displays a liver receptor binding affinity similar to that of wild type bGH but acts to suppress growth in transgenic mice, we proposed that the growth-promoting domain of GH is distinct from its receptor-binding region and that this analog acts as a functional antagonist (26-27). In order to further dissect the molecular bases of DTM, i.e. the importance of Glu 117, Gly 119, and Ala 122 in the growth-promoting activity of bGH, bGH analogs with single amino acid substitutions at position 117,119, or 122 were generated. The abilities of these analogs to bind to liver membrane preparations and to stimulate growth in transgenic mice were assessed.

Bovine GH (bGH) analogs with single amino acid substitutions at positions 117 (bGH-E117L), 119 (bGH-G119R), and 122 (bGH-A122D) were generated. These analogs bind to mouse liver membrane preparations with affinities similar to native bGH. However, transgenic mice which express the analogs demonstrate different phenotypes ranging from dwarfism to gigantism. For example, expression of bGH or bGH-E117L result in large transgenic mice. In contrast, transgenic mice with a growth phenotype similar to nontransgenic animals result from expression Of bGH-A122D. Surprisingly, transgenic mice with relatively high serum levels of bGH-G119R possessed a dwarf phenotype. Together these results suggest that Gly 119 and Ala 122 are involved in growth-promoting activity of GH. (Molecular Endocrinology 5: 1845-1852, 1991)

INTRODUCTION

GH is a member of a protein family which includes GH, PRL, and placental lactogen (PL). These genes are believed to have evolved from a common ancestral gene (1). GH isolated from various vertebrate species has been shown to possess highly conserved structural features (1-3). It is a single-chain polypeptide which contains 191 amino acids with a molecular mass of approximately 22,000 Daltons (4-7). Bovine GH (bGH) was isolated in 1944 (8). Since then a variety of GH genes and cDNAs have been cloned, including those derived from human (9), rat (10), cow (11), pig (11), and chicken (12). GH, as its name implies, regulates essential functions of animal growth (13). In addition to growth promotion, GH is also involved in regulating other metabolic processes, including lipid, nitrogen, mineral, and carbohy-

RESULTS In Vitro Mutagenesis

0888-8809/91/1845-1852S03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society

All mutated bGH genes were generated by an oligonucleotide-directed mutagenesis protocol. The mutations 1845

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 July 2015. at 07:26 For personal use only. No other uses without permission. . All rights reserved.

Vol5No. 12

MOL ENDO-1991 1846

resulted in the substitution of Leu for Glu at position 117, bGH-G117L; Asp or Thr for Ala at position 122, bGH-A122D or bGH-A122T; and Arg, Lys, Leu, Pro, or Trp for Gly at position 119, bGH-G119R, bGH-G119K, bGH-G119L, bGHG119P, or bGH-G119W, respectively. All of the mutations were confirmed by oligonucleotide sequence analyses (data not shown).

Table 1. Summary of Founder Transgenic Mice Which Express bGH Genes Encoding Single Amino Acid Substitutions in the Helix III Region bGH analogs

bGH-E117L bGH-G119R

Expression in Mouse L Cells Plasmids encoding the mutated and wild type (pBGH10A6) bGH genes were transiently introduced into cultured mouse L cells. Five days post transfection, culture fluids were analyzed for bGH by immunoblotting. The results are shown in Fig. 1. All plasmids direct expression of bGH, the majority of which was secreted and found in the culture media (Fig. 1).

bGH-A122D

a

Animal

4a 37a 25 28a 49a 53 94 138 33

Copy no. 1

2 1 25

Sex

F F M

Serum bGH Growth ratio (2 month)

0.4 2.0 0.5 0.9 6.0 1.5 0.2 3.0 10

1

M M M F F M

67a

1

M

10

95

1

132 a

1

M M

8.0 10

1

1 1

5

1.7 1.7 0.93 0.88 0.60 0.85 0.98 0.74 0.92 0.88 0.78 0.72

Mouse lines have been developed from these animals.

RRA Competitive receptor binding studies were carried out using male mouse liver membrane preparations and serum-free conditioned media from mouse L cells transfected with each of the mutated bGH genes (26). Mean dissociation constants were calculated using the computer program LIGAND from three different experiments each of which was carried out in triplicate (26). Student t tests indicated no significant differences in dissociation constant values between wild type bGH (3.76 ± 1.9 nM), bGH-E117L (2.62 ± 2.09 nM), bGHG119R (2.77 ± 2.06 nM), or bGH-A122D (3.36 ± 1.6 nM). Values are the mean ± SD. Transgenic Mouse Production A series of transgenic mouse lines was produced by standard microinjection techniques (Table 1). Transgenic offspring were identified by slot blot hybridization analysis (data not shown). Mouse lines were propa-

A B C D E F G H

I

J

Fig. 1. Immunoblot Analysis of the bGH Gene Expression by Mouse L Cells Lane A represents the culture media from mouse L cells transiently transfected with native bGH gene (pBGH10A6). Lane B represents the culture media from mock transfected mouse L cells. Lanes C-J represent culture media from mouse L cells transiently transfected with bGH mutated genes: pBGHG119R, pBGH-G119K, pBGH-G119L, pBGH-G119W, pBGH-G119P, pBGHE117L, pBGH-A122D, and pBGHA122T, respectively.

gated which contained bGH genes ranging from 1-30 copies (Table 1). Offspring were assayed for the transgenes and for the presence of serum GH or GH analogs (data not shown; 25-27). All mice which expressed the mutated bGH transgenes were monitored for growth rates (Fig. 2). The growth ratios of the mice are summarized in Table 2. Mice which express bGH-E117L grow at the rate similar to mice which express wild type bGH (i.e. approximately 1.7). Mice which express bGHG119R at relatively high serum levels result in animals with a DTM phenotype. In addition, four additional amino acid substitutions at position 119 of bGH resulted in DTM when these analogs were expressed at high levels (Table 3). The degree of suppression was correlated with serum levels of the bGH analog (Fig. 3). A single substitution mutation at position 122 (bGHA122D) also resulted in animals that were statistically smaller than nontransgenic littermates. The degree of growth suppression by bGH-A122D was not as severe as that seen in DTM or bGH-G119R transgenic mice even in the presence of high serum levels of this bGH analog (Table 2). However, transgenic mice expressing a bGH analog, bGH-A122T, which converted the amino acid residue from Ala found in bGH to Thr found in hGH, demonstrated the same potency in growth enhancement as wild type bGH (Table 3). Figure 4 demonstrated the typical phenotypes of the transgenic mice which express either of the three bGH analogs with single amino acid substitutions.

DISCUSSION The search for a growth-related active domain in GH dates to the early 1970s (28-31). It was found that short sequences, such as residue 96-133 of bGH, purified after partial tryptic digestion, still retained significant rat tibia bone growth-stimulating activity, whereas the segments 1-95 and 134-191 had much less activity (28-31). Subsequently, recombinant hor-


Production of Dwarf Transgenic Mice

1847

Male

Table 2. Growth Ratio (Transgenic Mice/Nontransgenic Littermates) Comparison between Transgenic Mice Which Express bGH Genes Encoding a Single Amino Acid Substitution

Control (n-19) bGH (n-5) bGH-A122D (n-12) bGH-E117L (n-6) bGH-G119R (n-12) bGH-MB (n-8) T

Mean growth ratio of TG mice Transgenes

n 1 2 3 4 Month Month Month Month

1',

©

Female Control (n-17) bGH (n-7) bGH-A122D (n-7) bGH-E117L (n-7) bGH-G119R (n-7) bGH-M8 (n-15)

Male bGH bGH-E117L bGH-G119R bGH-A122D Female bGH bGH-E117L bGH-G119R bGH-A122D

Overall growth ratio (TG/NTG)

5 6 12 12

1.38 1.32 0.67 0.84

1.56 1.59 0.67 0.84

1.61 1.63 0.65 0.79

1.64 1.68 0.65 0.86

1.56s 1.55" 0.66a 0.83"

7 7 7 7

1.44 1.43 0.64 0.91

1.68 1.71 0.62 0.85

1.69 1.68 0.65 0.84

1.70 1.65 0.66 0.89

1.63" 1.61" 0.66a 0.89*

a Significantly different from the nontransgenic littermates ( P < 0.001). " Significantly different from nontransgenic littermates or bGH-G119R transgenic mice (P < 0.05).

Table 3. Summary of Founder Transgenic Mice Which Express bGH Genes Encoding Single Amino Acid Substitutions at Position 119 and 122

Age (weeks) Fig. 2. Growth Rate Comparison between Male and Female Transgenic Mice Containing bGH, bGH-M8, bGH-E117L, bGHG119R, bGH-A122D, and Their Nontransgenic Littermates at Age 4, 8,12, and 16 Weeks after Birth Numbers of the experimental animals for each group are indicated (n). The body weights of the experimental animals are expressed as the mean ± SD.

mones were generated with human GH (hGH) 1-134 linked to hPL 141-191 or hPL 1-134 linked to hGH 141-191 through a Cys53-Cys165 disulfide bond (32). These recombinant hormones were subsequently evaluated for their immunoreactivities as well as receptorbinding properties. It was found that the recombinant hGH (1 -134)-hPL (141 -191) retains hGH immunological activity and full somatogenic receptor-binding ability but had little hPL activity. On the other hand, the recombinant hPL (1-134)-hGH (141-191), possessed mostly hPL immunological activity and lactogenic receptorbinding characteristics, with negligible hGH activity. These results strongly suggested that immunological and biological activities of the hormones were determined mostly by the NH2-terminal 1-134 portions. Recently, site-directed antisera generated by peptide immunization have been used to study the antigenicity of bGH. Interestingly, antisera against the bGH residue

bGH analogs

Animal

bGH-G119P bGH-G119K

10

1 1

12

1

9

Sex

18 2 26 2 23 bGH-G119L 20 27 30 1 bGH-G119W 16 bGH-A122T n = 5 1-20 n = 5 1-20

F M

Serum bGH Growth ratio (2 month)

2.0 0.5

M

0.4

F F F M M M F

4.0 5.0 6.5 0.5 8.0 0.5-10 0.5-10

0.81 0.84 0.95 0.78 0.59 0.81 1.0 0.64 1.6 1.7

120-140 and 134-154 significantly increased the growth-promoting activity of bGH in vivo (33). This observation has been suggested to occur as a result of protection of bGH from proteolytic enzymes (33). The information about functional domains of GH obtained through fragment experiments is limited, since the overall conformation of the protein has been altered. An alternative approach is to employ a site-specific mutagenesis protocol in which a single or few amino acids of interest are altered and then tested for biological effects. This approach has been used effectively in defining the binding domains of hGH (34, 35). It was reported that amino acid residues 10 (N-terminus) 54, 56, 58, 64, 68 (loop region), and 171, 172, 175 178, 182, and 185 (C-terminus) are likely involved in receptor binding (34, 35). By combining site-specific mutagenesis of the bGH gene with the in vivo assay of the ability of the bGH analogs to regulate growth of transgenic mice, we have found that a growth-promoting region of bGH is local-


Vol5No. 12

MOL ENDO-1991 1848

0.5

2

4

6

8

10

bGH in Serum [ug/ml] Fig. 3. Relationship Between Serum bGH Analog Concentrations Which Contain Different Single Amino Acid Substitutions at Position 119 and the Growth Ratio of 14 Founder Transgenic Mice The ordinate represents bGH analog concentration in serum. The abscissa represents the growth ratio of transgenic/nontransgenic mice at 30 days of age. The correlation coefficient (r = 0.80) is highly significant (P < 0.01).

Fig. 4. Representative Transgenic Mice (2-Month-Old Males) That Express Different bGH analogs From right, Transgenic mice containing bGHE117L, bGHA122D, which is similar to the size of a nontransgenic mouse, and bGH-G119R. All mice express approximately 3-5 of the bGH analogs.

ized in the third a-helix of the protein (26). This area is distinct from the receptor-binding regions (34, 35). Three amino acid substitutions (E117L, G119R, and A122D) in the third a-helix of bGH were shown to alter

the activities of bGH from that of an agonist to an antagonist (26, 27). In the present study, we have extended this observation. Several generalizations are noteworthy from our study. First, the substitution of Leu 117 for Glu does not affect GH receptor binding or the ability to enhance growth in transgenic mice. Thus, this analog retains the same activities as native bGH. Second, all substitutions at amino acid position 119 (bGH-G119X; X = R, K, L, P, W) result in analogs in which the ability to bind to GH receptor has been uncoupled from the ability to enhance growth in transgenic mice. When these analogs are present at relatively high levels in the serum of transgenic mice, a DTM phenotype results. Third, substitution of Asp for Ala at residue 122 results in a bGH analog which binds to GH receptor but does not enhance growth in transgenic mice. However, unlike bGH-G119X analogs, the ability to retard mouse growth at relatively high serum levels was diminished. Thus, it appears that amino acid substitutions at position 119 are more effective than those at position 122 in growth suppression when the analogs are expressed at similar levels in transgenic mice. However, the salient feature of bGH-A122D and bGH-G119X is the apparent uncoupling of the ability to bind GH receptor with that of enhancing growth in transgenic mice. A comparison of amino acid sequences between known GH molecules reveals that Ala 122 is conserved only among nonprimate mammals (3). In primates, Thr is substituted for Ala, while Lys or Leu are found in other vertebrates. This variation suggests tolerance of amino acid substitutions at this position or alternatively that the different amino acids might contribute to species specificity of the GH molecules. Since hGH is biologically active in nonprimate mammals but not vice versa, it is reasonable to predict that transgenic mice which express this bGH analog (bGH-A122T) would possess the same phenotype as animals which express wild type bGH transgenic mice. Our results are in agreement with this prediction (Table 3). Glu 117 is conserved in GHs from mammals as well as chickens (3). Since the substitution mutation at this position (bGH-E117L) showed no effect on bGH growth-promoting activity, we conclude that residue 117 of bGH is not likely to be directly involved in GH's growth-promoting activity. Gly 119 is conserved among all members of the GH family, including PRL and PL (3). Glycine is unique among amino acids in that it possesses a single hydrogen atom as a side chain. This small side chain has been suggested to increase molecule flexibility (36). Consequently, it is the least favored residue for the formation of a stable a-helical structure (36). The absolute conservation of this a-helix destabilizing amino acid within a strong a-helical-forming region implies a crucial role of the residue. According to computer-simulated bGH helix III structure, a cleft is located near the center of this a-helix due primarily to Gly 119 (Fig. 5). Ala 122 is at the same phase of the a-helix, which extends the cleft due to its


1849


Fig. 5. Three-Dimensional Models of bGH and the bGH Analog (bGH-GH9R) The models were generated using an Intergraph Computer Aided Design System coupled with the Biomolecular Design program, which was developed by the College of Engineering and Technology at Ohio University. Amino acid substitution mutation of Arg for Gly at position 119 is indicated. The color codes are as follows: blue, carbon; white, hydrogen; red, nitrogen; green, oxygen; yellow, sulfur.

relatively small side chain. We postulate that this cleft is important for the growth-promoting activity of the GH molecule. If this cleft, which may serve as a hinge-like structure, is important for growth-related biological activity of the molecule, then Gly may be the only residue tolerable at this position. Any other substitution presumably would decrease the flexibility of the molecule, resulting in altered biological activity. The amino acid residues chosen to substitute for Gly 119 in our study were representative of various amino acid groups including: Leu (hydrophobic, 2.8); Lys (hydrophilic and positive charged, 2.8); Trp (bulky hydrophobic, 3.8); and Pro (helix breaker with bulky side chain, 2.2). The numbers in parentheses indicates the relative volume increases of the amino acid side chains as compared to Gly after the substitution mutation at position 119 (37). On the other hand, the effects of mutations at position 122 cannot be explained simply by the size of the amino acid side chain. Substitution of Ala 122 to Thr does not have an effect on bGH growth-promoting activity in transgenic mice (Table 3). In this case Thr is approximately the same size as Asp (both are 1.3 times

larger than Ala). Therefore, the possible involvement of the negative-charged side chain of Asp in destroying the growth-promoting activity of bGH must be considered. This result further suggests that a limited number of amino acids are acceptable at position 122 (e.g. Ala, Thr). Further mutational analyses are required to demonstrate that position 122 in GHs is responsible for the nonreciprocal growth-promoting activity of primate and nonprimate GHs. In addition, if Gly plays a structural role, i.e. determining the functional conformation of GH, then other amino acids in this region may be important in dictating the biological activities of the molecule.

MATERIALS AND METHODS Plasmid Construction and Mutagenesis All mutated plasmids are derivatives of the parental plasmid, pBGH10A6, which contains the complete coding region of bGH and intron A. The bGH gene was ligated to a 1700-base pair segment of the mouse metallothionein I transcriptional regulatory sequence (Fig. 6). All mutations in the third a-helix of bGH were generated by


MOL ENDO-1991 1850

Vol5No. 12

EcoRI

amHI/Bgin ATG

XmaI

5" GT GTC TAT GAG AAG CTG AAG GAC CTG GAG GAA GGC ATC CTG GCC CTG ATG CGGGAG CTG GAAGAT GGC ACC CC 3'

109 110111 112 113 114 115 116 117 118 119 120121 122 123124 125126 Glu Gly Ala Leu Arg Asp Pro Thr Leu Trp Lvs

Fig. 6. General Strategy of Oligonucleotide Site-Directed Mutagenesis pBGH10A6 was used as the parental vector. It contains mouse metallothionein I transcriptional regulatory sequences fused to a bGH mini-gene which contains five exons (shaded boxes I-V) and intron A (26). This fusion gene was incorporated into pBR322 at the EcoRI site. The pBR322 origin of replication (ORI) as well as the ampicillin-resistant gene (Amp) are indicated. The nucleotide sequence between restriction sites Tth'\'\'\\ and XmaI is shown. The positions of corresponding amino acid residues are indicated along with the nucleotide sequence. Amino acid substitutions are also indicated.

oligonucleotide-directed mutagenesis (38) using complementary oligonucleotides to replace the DNA fragment between the ft/71111 site, found near the 3' end of Exon IV, and the XmaI site, located near the 5' end of Exon V. In addition, this oligonucleotide duplex (a plus strand 73 mer and a minus strand 76 mer) encoded a silent base pair change which created a unique SamHI restriction site (25, 26). The oligonucleotides were hybridized and subcloned between the Ttlr\'\'\\ and XmaI sites using standard procedures (39). All mutations were confirmed by sequencing. Cell Culture and Transient Expression Assay Mouse L cells were used for in vitro expression analyses as described (40). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Grand Island, NY) plus 10% Nu-Serum (Collaborative Research, Bedford, MA) and 50 nQl ml gentamicin. A modification of a previously described transfection procedure was employed (41). Briefly, 4 ^9 plasmid DNA were added to 1.0 ml DMEM containing 0.2 mg diethylaminoethyl (DEAE)-dextran. This solution was added to approximately 106 cells in a 35-mm tissue culture plate which had been washed previously with 2.0 ml DMEM. After incubation of the cells at 37 C for 60 min, the DNA-DEAE-dextran solution was removed and the cells shocked for 90 sec with 2.0 ml 5% dimethylsulfoxide in HEPES-buffered saline at room temperature. The solution was aspirated and cells washed with 2.0 ml DMEM. Media containing 10% Nu-Serum and 50 ^g/ml gentamicin were added and changed daily. Five days post transfection, culture fluids were collected for immunoblotting analyses. Polyacrylamide-Gel Electrophoresis and Immunoblotting Four microliters of mouse serum from Mt/bGH transgenic animals or 20 n\ culture media from day 5 posttransfected

mouse L cells were analyzed by 12.5% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membranes at 100 V constant voltage for 1 h (42). Immunoblot detection of bGH was performed by a modified procedure of Viceps-Morder (43). After protein transfer, the nitrocellulose paper was blocked with 2% gelatin in Trisbuffered saline (TBS) with gentle agitation for 1 hur at room temperature, and then washed three times with 0.05% Tween 20 in TBS (5 min per wash). Polyclonal rabbit anti-bGH (1:100 dilution) in 1 % gelatin/TBS was added to the nitrocellulose membrane and incubated overnight at room temperature with gentle agitation. After removing the primary antibody, the nitrocellulose paper was washed three times with 0.05% Tween 20 in TBS and subsequently incubated for 2 h at room temperature in the presence of a goat antirabbit immunoglobulin G horseradish peroxidase conjugate (Boehringer Mannheim Biochemicals, Indianapolis, IN) in 1 % Gelatin/TBS. After incubation with secondary antibody, the nitrocellulose was washed three times with 0.05% Tween 20 in TBS. To visualize the protein bands, the nitrocellulose paper was incubated for 10 min in a mixture of 50 ml 0.018% H2O2 (vol/ vol) in TBS and 10 ml methanol containing 30 mg horseradish peroxidase color development reagent (Bio-Rad, Richmond, CA). The nitrocellulose paper was then rinsed with water, airdried, and photographed. Purified bGH (a gift from Upjohn Co., Kalamazoo, Ml) was used to quantitate the expressed bGH level by photographic and densitometric methods (44). Radioreceptor Binding Assay Membrane binding studies were performed as previous described (26). Liver membrane preparations from C57BL/SJL hybrid mice of either sex (60-120 days old) were homogenized with a Brinkman Polytron homogenizer (Brinkman Instruments, Westbury, NY) in 4 vol (wt/vol) 0.3 M sucrose, 10 miui EDTA, 50 mM HEPES, 0.1 mM tosylphenylalanine chloromethylke-


1851


tone, and 1 mM phenlymethylsulfonyl fluoride at pH 8.0. The above steps and all the following protocols were carried out at 4 C. The homogenate was centrifuged at 20,000 x g for 30 min, and the supernatant was centrifuged at 100,000 x g for 1 h. The pellets were washed once with 10 mM HEPES, pH 8.0, and recentrifuged. These pellets were resuspended in 10 mM HEPES, pH 8.0, to a protein concentration of approximately 50 mg/ml. Samples of the membranes were frozen on solid CO2 and stored at - 2 0 C. Membrane protein concentrations were determined by BioRad protein assay. Competitive binding assays were performed using the following protocol. Microsomal membranes corresponding to 1 mg protein were incubated with [125l]hGH (0.5 ng/ml; SA, 100 MCi/^g) and with various amount of unlabeled bGH as well as bGH analogs ranging from 1-400 ng in a total vol of 0.3 ml assay buffer (20 mM HEPES, 10 mM CaCI2, 0.1% BSA, and 0.05% NaN3, pH 8.0). After a 2- to 3-h incubation at room temperature, the reaction was stopped by addition of 1 ml icecold assay buffer followed by centrifugation at 10,000 x g for 15 min. Membrane pellets then were assayed for radioactivity. All assays were performed either in duplicate or in triplicate and repeated three to four times. Transgenic Mouse Production The procedure for production of transgenic mice by direct microinjection of DNA into the male pronucleus of fertilized mouse eggs obtained from B6SJLF1/J (C57BL/6J x SJL/J) was described previously (45). Transgenic mouse lines were propagated by crossing founder mice with B6SJLF1/J nontransgenic individuals. DNA extraction from mouse tails and hybridization analyses (slot blot) were as described (25-27). Sera from hybridization-positive mice were tested and quantified for the presence of bGH by immunoblotting as described above.

Acknowledgments We thank Eric Holle and Diane Payne for excellent technical assistance. Rabbit anti-bGH sera was a gift from Dr. Fritz Rottman, Case Western Reserve University (Cleveland, OH).

Received August 19, 1991. Revision received October 3, 1991. Accepted October 3,1991. Address requests for reprints to: Dr. John J. Kopchik, Edison Animal Biotechnology Center, Department of Zoology, Molecular and Cellular Biology Program, Wilson Hall/West Green, Ohio University, Athens, Ohio 45701. This work was supported in part by the State of Ohio Eminent Scholar Program, which includes a grant by Milton and Lawrence Goll (to J.J.K.).

REFERENCES 1. Miller WL, Eberhardt NL 1983 Structure and evolution of the growth hormone gene family. Endocr Rev 4:97-129 2. Nicoll CS, Mayer GL, Russell SM 1986 Structural features of prolactins and growth hormones that can be related to their biological properties. Endocr Rev 7:169-203 3. Watahiki M, Yamamoto M, Yamakawa M, Tanaka M, Nakashima K 1989 Conserved and unique amino acid residues in the domains of the growth hormones. J Biol Chem 264:312-316 4. Andrews P 1966 Molecular weights of prolactin and pituitary growth hormones estimated by gel filtration. Nature 209:155-157 5. Dellacha JM, Santome JA, Faiferman L 1966 Molecular weight of bovine growth hormone. Experientia 22:16-17

6. Ellis GJ, Marler E, Chen HC, Wilhelm AE 1966 Molecular weight of bovine, porcine and human growth hormone by sedimentation equilibrium. Fed Proc 25:348 7. Miller WL, Martial JA, Baxter JD 1980 Molecular cloning of DNA complementary to bovine growth hormone mRNA. J Biol Chem 255:7521-7524 8. Li CH, Evans HM 1944 The isolation of pituitary growth hormone. Science 99:183-184 9. Goeddel DV, Heyneker HL, Hozumi T, Arentzen R, Itakura K, Yansura DG, Ross MJ, Miozzari G, Crea R, Seeburg PH 1979 Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 281:544-548 10. Seeburg PH, Shine J, Martial JA, Baxter JD, Goodman HM 1977 Nucleotide sequence and amplification in bacteria of the structural gene for rat growth hormone. Nature 270:486-494 11. Seeburg PH, Sias S, Adelman J, DeBoer HA, Hayflick J, Jhurani P, Goeddel DV, Heyneker HL 1983 Efficient bacterial expression of bovine and porcine growth hormones. DNA 2:37-45 12. Lamb JC, Galehouse DM, Foster DN 1988 Chicken growth hormone cDNA sequence. Nucleic Acids Res 16:9339 13. Isaksson OG, Eden S, Jansson JO 1985 Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol 47:483-499 14. Milman AE, Russell JA 1950 Some aspects of purified pituitary growth hormone on carbohydrate metabolism. Endocrinology 47:114-119 15. Swislocki Nl, Szego CM 1965 Acute reduction of plasma nonesterified fatty acid by growth hormone in hypophysectomized and Houssay rats. Endocrinology 76:665671 16. Swislocki Nl 1968 Effects of nutritional status and the pituitary on the acute plasma free fatty acid and glucose responses of rats to growth hormone administration. Metabolism 17:174-181 17. Swislocki Nl, Sonenberg M, Yamasaki N 1970 In vitro metabolic effects of bovine growth hormone fragment in adipose tissue. Endocrinology 87:900-904 18. Kostyo JL, Nutting DF 1973 Acute in vivo actions of growth hormone on protein synthesis in various tissues of the hypophysectomized rats and their relationship to the levels of thymidine factor and insulin in the plasma. Horm Metab Res 5:167-174 19. Goodman HM 1978 Effects of growth hormone on the utilization of L leucine in adipose tissue. Endocrinology 102:210-217 20. Goodman HM, Grichting C, Coiro V1986 Growth hormone action on adipocytes. In: Raiti S, Tolman RH (eds) Human Growth Hormone. Plenum Press, New York, pp 499-512 21. Press M 1988 Growth hormone and metabolism. Diabetes Metab Rev 4:391-414 22. Smal J, Closset J, Hennen G, DeMeyts P 1987 Receptor binding properties and insulin-like effects of human growth hormone and its 20K variant in rat adipocytes. J Biochem 262:11071-11079 23. Kostyo JL 1986 The multipotent nature of growth hormone. In: Raiti S, Tolman RH (eds) Human Growth Hormone. Plenum Press, New York, pp 449-454 24. Salem MAM 1988 Effects of the amino-terminal portion of human growth hormone on glucose clearance and metabolism in normal, diabetic, hypophysectomized, and diabetic-hypophysectomized rats. Endocrinology 123:15651576 25. Chen WY, Wight DC, Chen NY, Colman TC, Wagner TE, Kopchick JJ 1991 Mutations in the third a-helix of bovine growth hormone dramatically affect growth hormone secretion in vitro and growth enhancement in transgenic mice. J Biol Chem 266:2252-2258 26. Chen WY, Wight DC, Wagner TE, Kopchick JJ 1990 Expression of a mutated bovine growth hormone gene


MOL ENDO-1991 1852

27.

28.

29.

30. 31. 32.

33.

34.

35.

suppresses growth of transgenic mice. Proc Natl Acad Sci USA 87:5061-5065 Chen WY, White ME, Wagner TE, Kopchick JJ 1991 Functional antagonism between endogenous mouse growth hormone (GH) and a GH analog results in dwarf transgenic mice. Endocrinology 129:1402-1408 Yamasaki N, Kangawa K, Kobayashi S, Kikutani M, Sonenberg M 1972 Amino acid sequence of a biologically active fragment of bovine growth hormone. J Biol Chem 247:3874-3880 Yamasaki N, Shimanaka J, Sonenberg M 1975 Studies on the common active site of growth hormone: revision of the amino acid sequence of an active fragment of bovine growth hormone. J Biol Chem 250:2510-2514 Hara K, Chen CJH, Sonenberg M 1978 Recombination of the biologically active peptides from a tryptic digest of bovine growth hormone. Biochemistry 17:550-556 Hara K, Sonenberg M 1977 Polyalanylation of bovine somatotropin peptide 96-133. Biochim Biophys Acta 492:95-101 Russell J, Sherwood LM, Kowalski K, Schneider AB 1981 Recombinant hormones from fragments of human growth hormone and human placental lactogen. J Biol Chem 256:296-300 Aston R, Rathjen DA, Holder AT, Bender V, Trigg TE, Cowan K, Edwards J, Cowden WB 1991 Antigenic structure of bovine growth hormone: location of a growth enhancing region. Mol Immunol 28:41-50 Cunningham BC, Wells JA 1989 High-resolution epitope mapping of hGH receptor interaction by alanine-scanning mutagenesis. Science 244:1081-1085 Cunningham BC, Jhurani P, Ng P, Wells JA 1989 Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science 243:1330-1336

Vol5No. 12

36. Chou PY, Fasman DG 1978 Empirical predictions of protion conformation. Annu Rev Biochem 47:251-276 37. Stryer L 1988 Prelude. In: Stryer L (ed)Biochemistry. W. H. Freeman and Co., New York, pp 3-42 38. Vlasuk GP, Inouye S1983 Site-specific mutagenesis using synthetic oligodeoxyribonucleotides as mutagens. In: Inouye M (ed) Experimental Manipulation of Gene Expression. Academic Press, New York, pp 291-303 39. Maniatis T, Fritsch EF, Sambrook J 1984 Molecular Cloning—A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 40. Kopchick JJ, Malavarca RH, Livelli TJ, Leung FC 1985 Use of avian retroviral-bovine growth hormone DNA recombinants to direct expression of biologically active growth hormone by cultured fibroblasts. DNA 4:23-31 41. Lopata MA, Cleveland DW, Sollner WB 1984 High levels of transient expression of a chloramphenicol acetyl transferase gene by a DEAE dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 12:5707-5717 42. Towbin H, Stephelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 43. Viceps-Madore D, Cidlowski JA, Kittler JM, Thanass JW 1983 Preparation, characterization and use of monoclonal antibodies to vitamin B6. J Biol Chem 258:2689-2696 44. Fernandez E, Kopchick JJ 1990 Quantitative determination of growth hormone by immunoblotting. Anal Biochem 191:268-271 45. Wagner TE, Hoppe PC, Jollick JD, Scholl DR, Hodinka RL, Gault JB1981 Microinjection of a rabbit /3-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc Natl Acad Sci USA 78:63766380


Trinitrophenylation of bovine growth hormone.

Stimulatory effect of glycine on human growth hormone secretion.

Bovine growth hormone: human food safety evaluation.

Bovine growth hormone meets new safety concerns.

FDA publishes bovine growth hormone data.

Evidence that bovine growth hormone treatment increases the rate of extrathyroidal 5'-monodeiodinase activity in cattle.

Growth hormone is permissive for neoplastic colon growth.

Growth hormone and ovulation: is there an indication for treatment of infertile women with growth hormone?

Multiple Effects of Growth Hormone in the Body: Is it Really the Hormone for Growth?

Somatomedin-like effects of biologically active bovine growth hormone fragments.

Growth factor choice is critical for successful functionalization of nanoparticles.

Growth hormone-binding protein activity is inversely related to 24-hour growth hormone release in normal boys.

Enhancement of the growth promoting activity of human growth hormone.

Mechanosensing is critical for axon growth in the developing brain.

Ultrastructural localization of prolactin, growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary.

Insect growth regulators with juvenile hormone activity.

Ornithine decarboxylase activity is critical for cell transformation.

Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.

Effect of genetic background on growth of mice hemizygous for wild-type or dwarf mutated bovine growth hormone transgenes.

Role of selected endogenous peptides in growth hormone-releasing hexapeptide activity: analysis of growth hormone-releasing hormone, thyroid hormone-releasing hormone, and gonadotropin-releasing hormone.

Correction for Chesnokova et al., Growth hormone is permissive for neoplastic colon growth.

Exogenous recombinant bovine growth hormone stimulates growth and hepatic IGF expression in shovelnose sturgeon Scaphirhynchus platorhynchus.

A magnetosome-associated cytochrome MamP is critical for magnetite crystal growth during the exponential growth phase.

Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.