Biomedical subjects

J H Nilson

Publications and source records attributed to J H Nilson.

At least 55 records · Page 3Linked to original sources

PubMed1989

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.

Animals↗

PubMed1989

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer.

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.

Animals↗

PubMed1989

The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1988

Characterization of the cAMP responsive elements from the genes for the alpha-subunit of glycoprotein hormones and phosphoenolpyruvate carboxykinase (GTP). Conserved features of nuclear protein binding between tissues and species.

Cyclic AMP responsive elements (CRE) have been identified in several genes, including those encoding the alpha-subunit of glycoprotein hormones and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) from the rat. Common to the CRE in these genes is the palindromic sequence T(G/T)ACGTCA. Based upon the strong conservation of this element, we hypothesize that the CRE functions by binding a protein that has been conserved across tissue and species lines. Scatchard analysis of gel mobility shift assays indicate that a nuclear protein in extracts prepared from rat liver and from a human choriocarcinoma cell line binds with high affinity to the cAMP responsive element from either gene (Kd approximately 10(-10) M). In order to identify the critical nucleotides within the CRE from these two genes, a series of oligodeoxynucleotides containing systematic mutations was synthesized and tested for protein binding and transcriptional function. Mutations within the palindromic core of either CRE resulted in a marked loss of binding to the nuclear proteins. Sequences outside the 8-base pair element were less important for nuclear protein binding to the PEPCK CRE and were not important for the alpha-subunit CRE. The relative binding, as determined by gel shift assays, correlated with the ability to confer cAMP responsive transcription to a viral promoter in transfected choriocarcinoma cells. DNase I protection assays suggest that binding of the nuclear factor from rat liver to the PEPCK CRE is more efficient when the core sequence is present in the intact PEPCK promoter regulatory region as compared to the isolated CRE oligodeoxynucleotide. Collectively, these results indicate that the nuclear factors necessary for cAMP induction of transcription of the alpha-subunit and PEPCK genes are conserved between tissues and species. In addition to the conserved features of these cis- and trans-active elements, nonconserved sequences and other elements of the promoter regulatory region influence the affinity of the protein-DNA interaction.

Animals↗

PubMed1988

Cyclic AMP and phorbol esters interact synergistically to regulate expression of the chorionic gonadotropin genes.

Previous studies have shown that activators of the protein kinase A pathway increase transcription of the genes encoding the alpha- and beta-subunits of human chorionic gonadotropin (hCG) in choriocarcinoma cell lines. Here, we show that treatment of choriocarcinoma cells with activators of protein kinase C, such as phorbol myristate acetate (PMA) and dioctanoylglycerol, increases accumulation of the mRNAs for both subunits of hCG by 3-4-fold. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, has no effect on hCG mRNA levels. To test the possibility that these two major intracellular signaling pathways interact, we treated choriocarcinoma cells with PMA, forskolin, or PMA and forskolin together. Treatment with either agent led to a 2-3-fold increase in hCG mRNA levels, whereas treatment with both agents resulted in a 9-fold increase. This synergistic response also occurred when choriocarcinoma cells were treated with PMA and 8-Br-cAMP. Furthermore, PMA did not increase intracellular cAMP levels, suggesting that these two pathways interact subsequent to cAMP generation. PMA also increased transcription of the hCG alpha- and beta-genes by 2-3-fold. Whereas transcription of the alpha subunit gene increases synergistically after treatment with both PMA and forskolin, transcription of the hCG beta-gene was limited to the increase caused by either agent alone. This latter result suggests that regulation of hCG beta mRNA accumulation is more complex than that of alpha-subunit mRNA and probably involves both transcriptional and post-transcriptional components.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1988

Methotrexate-induced amplification of the bovine lutropin genes in Chinese hamster ovary cells. Relative concentration of the alpha and beta subunits determines the extent of heterodimer assembly.

Methotrexate-induced gene amplification increased the expression of biologically active bovine luteinizing hormone (bLH) approximately 11-fold after stable transfection of a line of Chinese hamster ovary cells with genes encoding dihydrofolate reductase and the alpha and beta subunits of bLH. Subsequent analysis of the bovine genes revealed that while the alpha gene was amplified in response to methotrexate selection, the LH beta subunit gene remained unaffected. This effect was probably due to the linkage of the alpha subunit gene with the dihydrofolate reductase gene, the selectable and methotrexate-sensitive marker in the plasmid construct. Prior to methotrexate selection, the concentration of LH beta mRNA and the rate of LH beta synthesis exceeded that of alpha subunit mRNA and protein. Stepwise selection with methotrexate led to a progressive increase in the synthesis and secretion of biologically active bLH. Enhanced production of bLH correlated directly with similar increases in both the steady-state level of alpha subunit mRNA and the relative synthesis rate of alpha subunit protein. Despite progressive changes in alpha subunit concentration, formation of the alpha/beta heterodimer was always incomplete, even when the concentration of alpha subunit exceeded that of LH beta. Cumulatively, these results are consistent with a model in which the extent of steady-state combination of the subunits is determined by the mutual affinity and concentration of both subunits within the lumen of the secretory pathway. This stands in contrast to the long held view that the extent of glycoprotein hormone assembly is limited by the concentration of the beta subunit.

Animals↗

PubMed1987

Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element.

cAMP regulates transcription of the gene encoding the alpha-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, we inserted fragments from the 5' flanking region of the alpha-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between positions -146 and -111. In the absence of cAMP, the alpha-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. We localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the alpha-subunit gene.

Base Sequence↗

PubMed1987

Cyclic AMP regulates transcription of the genes encoding human chorionic gonadotropin with different kinetics.

Choriocarcinoma cell lines characteristically synthesize and secrete the alpha- and beta-subunits of human chorionic gonadotropin (hCG), as well as the intact heterodimer. Treatment of one such cell line, BeWo, with 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP) causes at least a 10-fold increase in the concentration of the mRNA encoding each subunit. Changes in mRNA concentrations are associated with similar changes in transcription rates of both the CG alpha and CG beta genes, although the kinetics of their transcriptional responses are different. Transcription of the alpha-subunit gene increases rapidly and becomes maximal within 1 hr after addition of 8-Br-cAMP. By contrast, transcription of the CG beta gene increases slowly and progressively for at least 8 hr after treatment with 8-Br-cAMP. The slow transcriptional response of the CG beta gene(s) appears to be unique compared to that of other cAMP-responsive genes, and suggests that the cyclic nucleotide may regulate transcription of the CG genes by different mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

Expression of the genes encoding bovine LH in a line of Chinese hamster ovary cells.

Synthesis of biologically active LH is complex, due in part to its heterodimeric subunit structure and to the numerous post-translation modifications of each subunit. Through the use of mammalian expression vectors we have been able to introduce the bovine alpha subunit and LH-beta genes into a Chinese hamster ovary cell line deficient in dihydrofolate reductase. The bovine genes are actively expressed and the Chinese hamster ovary cells secrete biologically active LH. The expression vector containing the bovine alpha subunit gene also contains a modified mouse gene encoding dihydrofolate reductase, permitting the use of methotrexate to amplify selectively the bovine alpha subunit gene after its integration into the genome of the Chinese hamster cells. This provides a novel means for assessing the importance of alpha subunit concentration with respect to assembly of the heterodimer. In addition, methotrexate selection leads to the over-production of LH (10 micrograms/10(6) cells/24 h). Finally, because the bovine LH produced in the Chinese hamster ovary cells is glycosylated, this transfection system can be used in conjunction with in-vitro mutagenesis to determine whether site-specific changes in glycosylation have an effect on subunit assembly and biological activity. This transfection approach therefore offers multiple avenues to explore further the molecular mechanisms underlying the complex biosynthetic pathway of bovine LH.

Animals↗

PubMed1986

Measurement of messenger ribonucleic acid for luteinizing hormone beta-subunit, alpha-subunit, growth hormone, and prolactin after hypothalamic pituitary disconnection in ovariectomized ewes.

A transnasal, transsphenoidal surgical approach was used to perform hypothalamic pituitary disconnections (HPD) in ovariectomized (OVX) ewes to examine the role of the hypothalamus in regulating the synthesis of anterior pituitary hormones. Ewes were killed at 1-3 days (n = 6), 1 week (n = 5), or 1 month (n = 5) after HPD. Pituitary glands were removed, and hemisected for analysis of hormone or messenger RNA (mRNA) content. Blot hybridization using specific complementary DNA probes was used to quantify the concentration of mRNA for LH beta-subunit, alpha-subunit, GH, and PRL. Concentrations of mRNA for LH beta- and alpha-subunits were lower (P less than 0.01) at 1-3 days after HPD than in OVX ewes. At 1 week and 1 month after HPD, concentrations of mRNA for LH beta- and alpha-subunits were near the lower limit of detection of this assay system. In contrast, for 30 days after HPD, pituitary concentrations of mRNA for GH and PRL were not different (P greater than 0.05) from those in OVX ewes. At 1 week and 1 month after HPD, pituitary content of LH, FSH, and GH was lower (P less than 0.01) than in OXV ewes. Pituitary PRL content in all HPD ewes was lower (P less than 0.05) than in OVX ewes. In a separate group of five ewes that were bled daily for 30 days after HPD, serum concentrations of LH and FSH fell dramatically during the first 3 days after HPD. In contrast, serum concentrations of GH and PRL remained similar to pre-HPD concentrations for 30 days after HPD. Thus, hypothalamic stimulation is essential for maintaining the concentration of mRNA for LH beta- and alpha-subunits within the anterior pituitary gland. Without continued hypothalamic support, pituitary and serum concentrations of LH and FSH rapidly decline. In contrast, concentrations of mRNA for GH and PRL are maintained in the absence of hypothalamic input.

Animals↗

PubMed1985

The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5'-untranslated region.

Both cDNA and genomic clones encoding the beta subunit of bovine luteinizing hormone (LH) have been isolated and characterized. The nucleotide sequence was determined for the entire gene and 776 base pairs of 5'-flanking sequence. The mRNA cap site and polyadenylation site were mapped by primer extension and S1 nuclease protection, respectively. The bovine LH beta spans less than 1.1 kilobase pairs and has three exons encoding a 550 nucleotide mRNA (excluding the poly(A) tail). Bovine LH beta is a single-copy gene, in contrast to human LH beta, which is a member of the LH/chorionic gonadotropin beta subunit multigene family. Comparison of the bovine LH beta gene with the human LH beta/chorionic gonadotropin gene family reveals a high degree of nucleotide sequence homology, both within the genes and in the 5'-flanking sequences. Despite this extensive sequence conservation, there is a major difference between the two species in the selection of a promoter site. As a result, the bovine LH beta gene produces an mRNA with an usually short 5'-untranslated region of only 6-11 nucleotides.

Amino Acid Sequence↗

PubMed1985

The common alpha subunit of bovine glycoprotein hormones: limited formation of native structure by the totally nonglycosylated polypeptide chain.

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Animals↗

PubMed1985

Expression of biologically active bovine luteinizing hormone in Chinese hamster ovary cells.

Biologically active bovine luteinizing hormone (LH) has been obtained through expression of the alpha- and LH beta-subunit genes in stably transformed clones of DUXB11, a Chinese hamster ovary cell line deficient in dihydrofolate reductase (DHFR). Expression of alpha-and LH beta-subunit mRNAs of the expected sizes (approximately 910 and 770 nucleotides, respectively) were revealed by blot analysis after electrophoresis of total cellular RNA. Furthermore, presence or absence of the gonadotropin mRNAs in several clonal lines was directly correlated with the appearance of one or both bovine LH subunits in the culture medium. Media from three clones secreting significant immunoreactive levels of both subunits also stimulated the release of progesterone in ovine luteal cells, suggesting that the secreted LH was assembled into a biologically active and glycosylated dimer. Immunoprecipitation and NaDodSO4/PAGE of [35S]methionine-labeled proteins secreted from one of the clones, CHODLH20, further confirmed the presence of an alpha/beta dimer with apparent subunit molecular weights of 20,500 and 16,000, only slightly higher than those of pituitary alpha and LH beta subunits.

Animals↗

PubMed1985

Measurement of messenger RNA for luteinizing hormone beta-subunit and alpha-subunit during gestation and the postpartum period in ewes.

Pituitary content of luteinizing hormone (LH) and mRNAs for LH beta-subunit (LH beta), alpha-subunit, prolactin, and growth hormone were measured in ewes on Days 50 and 140 of gestation and on Days 2, 13, 22, and 35 postpartum. Content of LH in dissociated anterior pituitary cells declined (P less than 0.05) between Days 50 and 140 of gestation and remained low at 2 days postpartum. By 22 days postpartum, pituitary concentrations of LH were comparable to concentrations in normally cycling ewes. During gestation concentrations of mRNA for LH beta and alpha-subunit paralleled changes in cellular content of LH, reaching minimal levels on Day 140. By Day 2 postpartum, pituitary concentrations of mRNAs for LH beta and alpha-subunit began to increase; they reached maximum levels by Day 13 postpartum. There appeared to be a gradual linear increase in mRNA for prolactin through gestation and the postpartum period. No changes in mRNA for growth hormone were noted during the prepartum or postpartum periods. These data suggest that the decline in pituitary concentrations of LH during gestation is due to a decrease in cellular mRNA for LH beta and alpha-subunit. The increase in mRNA for LH beta and alpha-subunit appears to precede an increase in cellular content of LH in the postpartum ewe by several days.

Animals↗

PubMed1985

Coordinate regulation of the messenger ribonucleic acids encoding the alpha- and beta-subunits of human chorionic gonadotropin in HeLa cells and butyrate-resistant variants.

Short term exposure of HeLa S3 cells to sodium butyrate induces accumulation of the mRNAs encoding both the alpha- and beta-subunits of hCG. Both mRNAs accumulate with the same kinetics and reach maximal levels with the same concentration of butyrate, suggesting that the levels of alpha and hCG beta mRNAs are coordinately regulated. In addition, induction of both mRNAs is tightly coupled with a similar increase in secreted levels of hCG subunit protein, further suggesting that regulation of CG biosynthesis in HeLa cells occurs before translation. To determine whether HeLa cells which have overcome the growth inhibitory properties of butyrate show uniformly high levels of alpha and hCG beta mRNAs, we isolated clonal variants by stepwise selection with increasing concentrations of butyrate. Of 69 isolated variants, at least 2 (AO and B2) display different patterns of CG gene expression. In AO cells, alpha-subunit mRNA was not detectable, while in B2 cells, the level of alpha-subunit mRNA was similar to that of wild-type HeLa S3 cells. Conversely, hCG beta mRNA levels in both variant cell lines approximated levels found in butyrate-treated HeLa S3 cultures, suggesting that the mRNAs for alpha and hCG beta are regulated independently. Analysis of genomic DNA by blot hybridization indicated that the alpha-subunit gene was present as a single unrearranged copy in HeLa S3 cells and in both variants, indicating that differences in alpha-subunit gene expression are not associated with major genomic alterations, but probably reflect more subtle changes.

Butyrates↗

PubMed1983

Expression of alpha subunit and luteinizing hormone beta genes in the ovine anterior pituitary. Estradiol suppresses accumulation of mRNAS for both alpha subunit and luteinizing hormone beta.

Bovine cDNA clones containing coding sequences for growth hormone, prolactin, alpha subunit, and luteinizing hormone beta (LH beta) have been used to quantitate their respective mRNA concentrations in anterior pituitary glands isolated from ovariectomized ewes, or from ovariectomized ewes treated for three weeks with estradiol. Concentrations of mRNAs for prolactin or growth hormone remained unchanged in either physiological state. In contrast, treatment with estradiol resulted in a 98% decrease of mRNA for LH beta, relative to untreated animals. This change in mRNA was associated with a similar decrease in the concentrations of pituitary and serum LH. Administration of estradiol also led to a reduction (86%) of alpha subunit mRNA. These results suggest that estrogen regulates the expression of the genes encoding both the alpha and LH beta subunit prior to translation. Furthermore, the pronounced effect of estradiol on the concentrations of mRNAs for alpha subunit and LH beta suggest that the assembly of mature glycoprotein hormones may not be limited solely by the rate of accumulation of the beta subunit.

Animals↗

PubMed1983

Characterization and nucleotide sequence of the gene for the common alpha subunit of the bovine pituitary glycoprotein hormones.

The gene coding for the common alpha subunit of the bovine pituitary glycoprotein hormones was isolated from a bovine genomic library. The gene spans roughly 16.5 kbp, contains three intervening sequences, and codes for a message of approximately 730 nucleotides. The complete coding region of the gene was sequenced as well as 315 nucleotides of 5' flanking sequence and the entire intron C. Only a single base difference was found when the sequence of the gene was compared with that of the cDNA. Genomic blotting experiments suggest the presence of a single alpha subunit gene. Comparison of the bovine and human alpha subunit genes indicated that the high level of homology observed in the coding regions has been maintained throughout the 5' and 3' untranslated regions, and at least 90 nucleotides of the 5'flanking regions. Additionally, there is an 18 base pair sequence present in both the 5' flanking and 5' untranslated regions of the gene that is homologous to a region of the chick ovalbumin gene. This ovalbumin sequence has been suggested as a binding site for the progesterone receptor-complex.

Amino Acid Sequence↗

PubMed1983

Nucleotide sequence of a cDNA for the common alpha subunit of the bovine pituitary glycoprotein hormones. Conservation of nucleotides in the 3'-untranslated region of bovine and human pre-alpha subunit mRNAs.

A cDNA clone for the pre-alpha subunit of the pituitary glycoprotein hormones has been isolated from a bovine pituitary cDNA library through the use of a pool of synthetic oligodeoxynucleotide probes. This clone, designated pB alpha, contains a 564-base pair insert which includes a portion of the signal sequence, the entire coding sequence of the mature protein, and 224 base pairs of the 3'-untranslated sequence. As expected, the nucleotide and amino acid sequence of the mature bovine alpha subunit was homologous to the sequences reported for humans and rodents, with the most extensive homology occurring between bovine and rodents (85-90%). However, a comparison of the 3'-untranslated regions of pre-alpha subunit mRNA from three different mammalian species indicated that in bovine and rat, or in human and rat, these sequences have rapidly diverged, yielding respective homologies of 21 and 36%. In contrast, the sequence homology observed between the 3'-untranslated regions of bovine and human was 79%, which approaches the level of homology shared by their coding sequences. Thus, the conservation of the 3'-untranslated sequence in bovine and human pre-alpha subunit mRNA may be an indication that this region is functionally significant in these two species.

Amino Acid Sequence↗