Biomedical subjects

J H Nilson

Publications and source records attributed to J H Nilson.

At least 37 records · Page 2Linked to original sources

PubMed1995

Different composite regulatory elements direct expression of the human alpha subunit gene to pituitary and placenta.

To identify elements of the human alpha subunit gene necessary for cell-specific expression, we generated an array of block mutations spanning approximately 400 base pairs (bp) of promoter proximal region and examined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines. Comparison of promoter activity in the two cell types revealed both common and unique elements required for transcription in pituitary and placenta. Two strong elements, the cyclic AMP response element (CRE) and the upstream regulatory element (URE), regulate expression of the alpha subunit gene in BeWo cells. In contrast, promoter activity in alpha T3 cells requires an array of weaker elements. These include the CREs, the URE, as well as two previously described elements, pituitary glycoprotein hormone basal element (PGBE) and gonadotrope-specific element (GSE), and two new elements we designated as the alpha basal elements 1 and 2 (alpha BE1 and alpha BE2). These new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human alpha subunit promoter and bind distinct proteins designated alpha BP1 and alpha BP2, respectively. Southwestern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins. Additional studies disclosed several potential interactions between proteins that bind the CRE and proteins that occupy PGBE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific expression occurs through a unique composite regulatory element that includes components of the placenta-specific enhancer.

Animals↗

PubMed1995

Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility.

Animals↗

PubMed1995

The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus.

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation, androgens and Müllerian-inhibiting substance, and is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene had normal numbers and location of GnRH neurons but exhibited grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restored pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction.

Animals↗

PubMed1994

The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.

Animals↗

PubMed1994

Functional analysis of the placenta-specific enhancer of the human glycoprotein hormone alpha subunit gene. Emergence of a new element.

Placental expression of the alpha subunit gene of the human glycoprotein hormones requires a multicomponent enhancer composed of tandem cAMP-response elements and an adjacent upstream regulatory element. Based on recent studies indicating that the upstream regulatory element includes binding sites for more than one protein, we investigated how functional activity correlated with these binding sites. Through extensive replacement mutagenesis of the native promoter regulatory region, we provide the first functional map of the upstream regulatory element. Within this region, we find that distinct proteins interact with three overlapping binding sites. While each site is functionally significant, no single site is essential or displays clear dominance. This is surprising since one of the sites binds a placenta-specific protein that heretofore has been regarded as essential for activity of the human alpha subunit placenta-specific enhancer. Consequently, our refined functional map of the upstream regulatory element reveals a complex combinatorial code that directs expression of the human alpha subunit gene to placenta.

Animals↗

PubMed1994

Expression of the beta subunit of chorionic gonadotropin in transgenic mice.

Transcriptional activation of the chorionic gonadotropin (CG) genes is linked to trophoblast differentiation. In a multistep process, cytotrophoblasts expressing only the alpha subunit differentiate into intermediates that coexpress the CG beta subunit. To study the regulation of expression of the CG beta genes in vivo, we constructed mice carrying a 36-kilobase cosmid insert containing the six CG beta genes. In the placenta of all three constructed lines, expression occurred at approximately 1% of the levels in first trimester human placenta. The amount of CG beta mRNA in mouse placenta was a function of gestational age; however, in contrast to the human placenta where CG beta peaks early in pregnancy, CG beta transcripts were only detectable in the mouse placenta late in gestation, i.e. from day 14 onward. Human CG beta was expressed also in cerebral cortex, pituitary, and at minute levels in adrenal. Pituitary CG beta expression was significantly lower than in placenta. Unexpectedly, transcripts were observed in cerebral cortex at levels comparable with the placenta. Most of the CG beta transcripts in mouse placenta are derived from CG beta genes 5, 3, and 8, in a ratio similar to that found in human placenta. In contrast, only CG beta genes 1 and 2 were transcribed in transgenic mouse brain; open reading frames from the CG beta 1 and beta 2 transcripts differ substantially from the CG beta protein. The data show that although the mouse lacks a CG beta-like gene, the human CG beta genes are transcribed in a regulated fashion in mouse placenta. Moreover, the stage-specific induction of the transgene suggests that mouse placental cells may express CG beta in an intermediate cell comparable with that seen in human placenta. Taken together, these data suggest that transgenic mice can be used as a model for elucidating the mechanisms involved in regulated expression of the CG beta gene cluster in vivo. Additionally, a different subset of CG beta genes (CG beta 1 and beta 2) is active in the mouse brain.

Animals↗

PubMed1993

Transcriptional repression of the glycoprotein hormone alpha subunit gene by androgen may involve direct binding of androgen receptor to the proximal promoter.

Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.

Androgens↗

PubMed1992

Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.

Animals↗

PubMed1992

A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.

Amino Acid Sequence↗

PubMed1991

Different combinations of regulatory elements may explain why placenta-specific expression of the glycoprotein hormone alpha-subunit gene occurs only in primates and horses.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses.

Animals↗

PubMed1991

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.

Adrenocorticotropic Hormone↗

PubMed1991

Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor.

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.

Animals↗

PubMed1990

A cis-acting element located between the cAMP response elements and CCAAT box augments cell-specific expression of the glycoprotein hormone alpha subunit gene.

Several cis-acting elements interact through their cognate trans-acting factors to confer placenta-specific expression to the alpha subunit gene of glycoprotein hormones. These elements include two tandemly arranged cAMP response elements (CREs, located between base pairs (bp) -146 and -111 relative to the transcription start site), an upstream regulatory element (URE, located between -180 and -150), and a CCAAT box (located between bp -100 and -80). Here, we identify a new regulatory region, junctional regulatory element (JRE), with critical nucleotides located between the CREs and CCAAT box (bp -120 to -100). Although the binding site of the JRE abuts the 3' CRE, methylation interference assays indicate that no overlap occurs between the contact points of the proteins that bind to the JRE and CRE. This new regulatory element is highly conserved across species and contains a palindromic binding site for a 50-kDa nuclear factor(s) which is distinct from the factors that bind the URE, CRE, and CCAAT box of the alpha subunit gene. Finally, gene transfer studies suggest that, although JRE binding activity is found in several cell types, this element acts relatively cell-specifically. We conclude that this element and its cognate trans-acting factor act in conjunction with the complexes formed over the URE, CREs, and CCAAT box to enhance the placenta-specific activity of the alpha subunit promoter.

Base Sequence↗

PubMed1990

Complete and partial glycophospholipid anchors are found on a fusion protein consisting of luteinizing hormone beta subunit followed by a carboxyl-terminal domain of Thy-1.

The Thy-1 antigen is anchored to the cell surface by a carboxyl-terminal glycophospholipid moiety. To investigate the extent of anchor addition which occurs when such proteins cannot move efficiently to the cell surface, we have expressed a recombinant fusion protein composed of 107 amino-terminal amino acids of bovine luteinizing hormone beta subunit and 46 COOH-terminal amino acids of murine Thy-1 (Thy-1.2 allele). Although the limited amount of fusion protein transported to the cell surface is glycophospholipid-anchored, most of the protein accumulates in an intracellular, endoglycosidase H-sensitive form. The intracellular protein has an unusual structure that contains ethanolamine but does not bind detergent, suggesting either that anchor addition proceeds via a hydrophilic partial intermediate, or that anchor-degradative enzymes exist along the secretory path.

Animals↗

PubMed1990

The human alpha subunit glycoprotein hormone gene utilizes a unique CCAAT binding factor.

Placenta-specific expression of the gene encoding the alpha subunit of glycoprotein hormones involves the interaction of at least two different cis-acting elements: the upstream regulatory element (URE), which binds a placenta-specific trans-acting factor; and two tandem cAMP-response elements (CREs), which bind a ubiquitous protein (CRE-binding protein). To identify additional elements required for promoter activity, we used block replacement mutagenesis to produce a series of 10-base pair transversion mutations throughout the proximal promoter-regulatory region of the human alpha subunit gene. Transient expression of these constructs in choriocarcinoma cells enabled the identification of several closely spaced transcriptional control elements in addition to the previously identified URE and CREs. Gel mobility shift assays, methylation interference analyses, and UV cross-linking permitted identification of a factor that binds specifically to a canonical CCAAT box contained within the proximal promoter-regulatory of the human alpha subunit gene. This factor, which we refer to as alpha subunit CCAAT-binding factor (alpha CBF), is found in a variety of cell types and appears to be distinct from the previously characterized CCAAT-binding factors CTF/NF1, C/EBP, CP1, and NF-Y. We suggest that interaction of alpha CBF with factors binding to the URE and CREs may be required for maximal activity of the alpha subunit promoter in placental cells.

Base Sequence↗

PubMed1990

Different combinations of regulatory elements may account for expression of the glycoprotein hormone alpha-subunit gene in primate and horse placenta.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.

Animals↗

PubMed1990

Amplification of the transcriptional signal mediated by the tandem cAMP response elements of the glycoprotein hormone alpha-subunit gene occurs through several distinct mechanisms.

cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

CRE-binding proteins interact cooperatively to enhance placental-specific expression of the glycoprotein hormone alpha-subunit gene.

The 18-bp direct repeat occurring between positions -146 and -111 in the 5'-flanking region of the human alpha-subunit gene serves two functions: it mediates the transcriptional effect of cAMP and it acts in conjunction with an adjacent cis-acting element (URE) to confer properties of placental-specific expression to the alpha-subunit promoter. Functional activity of the URE and CRE requires binding of a trans-acting factor; each element binds a different factor. Analysis of saturation isotherms provides good evidence that cooperativity is involved in binding of CREB to the 18-bp direct repeat. This cooperativity could account for the synergistic effect of two CRE on both basal and cAMP-stimulated transcription. It remains to be determined whether heterotropic cooperativity is involved in binding of trans-acting factors to the URE and CRE. A major difference between the 5'-flanking region of the human alpha-subunit gene and comparable regions from bovine, rat, and mouse alpha-subunit genes is that the latter contain a single CRE homolog which appears incapable of binding the trans-acting factor that binds to the human alpha CRE. Lack of a functional CRE provides at least one explanation for inactivity of the bovine alpha-subunit promoter in choriocarcinoma cells and probably in bovine placenta as well. Yet, the same bovine promoter-regulatory region that lacks a functional CRE is capable of conferring pituitary-specific expression to the CAT gene in transgenic mice (data not shown). This suggests that the CRE is not required for pituitary-specific expression of the bovine alpha-subunit gene. Instead, another cis-acting element(s) must confer this property to the alpha-subunit promoter. While it is tempting to suggest that bovine, rat, and mouse alpha-subunit genes are not regulated by cAMP because of their inactive CRE homolog, it is also quite possible that other CRE are located further upstream. Accordingly, it will be of interest to obtain additional 5'-flanking sequence and determine whether functional homologs of the human alpha CRE are present in the bovine, rat, and mouse alpha-subunit genes, or whether another class of cis-acting elements provide cAMP-responsiveness.

Base Sequence↗