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The 5'-Flanking Region of the Ovarian Promoter of the Bovine CYP19 Gene Contains a Deletion in a Cyclic Adenosine 3',5'-Monophosphate-Like Responsive Sequence¹

Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry, Obstetrics, and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051

Address all correspondence and requests for reprints to: Evan R. Simpson, Ph.D., Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051.

	Abstract

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Conversion of C₁₉ steroids to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). In the ovary, P450arom is expressed in granulosa cells of both human (h) and bovine (b) follicles. After the ovulatory surge of gonadotropins, however, P450arom expression is maintained only in the luteinized granulosa cells of the human ovary and is absent from the bovine corpus luteum. We compared the regulation of expression of the ovary-specific human CYP19 (hCYP19_ov) and the bovine CYP19 (bCYP19_ov) gene by cAMP (forskolin) and sought to determine whether the divergence in the expression of P450arom with the onset of luteinization could be explained by specific cis-acting elements present uniquely in the 5'-flanking DNA of the hCYP19_ov or bCYP19_ov gene. We, therefore, subcloned DNA encompassing the promoters and 5'-flanking regions of the hCYP19_ov or bCYP19_ov gene into a promoterless luciferase vector. These constructs were transfected into luteinized bovine granulosa cells or bovine luteal cells in primary culture. Neither cell type exhibits endogenous expression of bovine P450arom. After transfection, cells were treated with either vehicle or 25 µM forskolin. There was little or no increase in luciferase activity after forskolin treatment in cells transfected with any of the bCYP19_ov constructs, whereas all of the corresponding hCYP19_ov constructs (-693/-16 to -214/-16 bp) expressed reporter activity in the presence of forskolin. This dramatic difference between the activities of the constructs of the two species occurred despite the fact that there is an 88% sequence identity between the bovine and human promoters in the region between -214 to -16 bp. One possible explanation for this variability may be that the bCYP19_ov gene has a 1-bp deletion in a cAMP-response element-like sequence (CLS) present at -208 to -201 bp in the hCYP19_ov gene that we have shown to be critical for cAMP-stimulated transcription of hCYP19_ov in the ovary. When this region of the bCYP19_ov promoter was mutated to the hCLS, a partial restoration in luciferase activity was observed after forskolin treatment. Therefore, these results suggest that another sequence in this -214 bp region of the bCYP19_ov gene is also contributing to the lack of expression of P450arom after luteinization in the bovine ovary. This lack of expression of the bCYP19_ov gene may be due to the presence of a repressive trans-acting factor expressed with the onset of luteinization of the bovine granulosa cell. These results further suggest that in the cow, elements upstream of those employed by the hCYP19_ov gene may have been recruited to facilitate regulated expression of the bCYP19_ov gene in the absence of a functional CLS.

	Introduction

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ESTROGENS play an important role in the development of various dimorphic anatomical, functional, and behavioral characteristics of males and females that are vital for reproduction of the species. To further our understanding of these actions of estrogens, it is important to gain insight into the regulation of estrogen biosynthesis. The conversion of C₁₉ steroids to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). It appears that, at least in the human, there is only one gene that encodes P450arom (1). This enzyme is localized in the endoplasmic reticulum of cells in which it is expressed (2) and uses the ubiquitous flavoprotein, NADPH-P450 reductase, to provide reducing equivalents derived from NADPH (2, 3, 4). P450arom expression is restricted to the gonads and brain in many species; however, in the human, a broader tissue distribution exists that includes the syncytiotrophoblast of the placenta (5), adipose tissue (6), bone (7), and various fetal tissues (8). One means by which tissue-specific expression of P450arom occurs in the human is through the use of alternative transcriptional start sites that arise as a result of the use of tissue-specific promoters. Expression of P450arom in the placenta relies primarily on a promoter at least 40 kb upstream from the start site of translation (9), while the promoter driving transcription in the ovary lies just upstream from the start site of translation (10). In adipose stromal cells and tissue, up to three alternative promoters may be employed (11).

To learn more about the regulation of expression of P450arom, we characterized the CYP19 gene in cattle. We chose to study this gene in cattle, because, like humans, cattle are a species that is monovulatory and exhibits placental formation of estrogens. We found that the bovine CYP19 gene, like the human gene, appears to use a promoter just upstream of the start site of translation to drive transcription in the ovary, whereas a promoter more distal drives transcription in the placenta (12). It has been well documented in a number of species that the granulosa cells of the ovarian follicle will synthesize 17ß-estradiol in response to FSH. Binding of FSH to its receptor on the granulosa cell results in an elevation of intracellular cAMP levels, which, in turn, stimulates the expression of P450arom in both human (13) and rat (14, 15) ovary. After the surge of gonadotropins that initiates the process of ovulation and luteinization, levels of 17ß-estradiol show a transient decline and then rise during the luteal phase in women (16, 17) or during pregnancy in rats (14, 15). This increase in 17ß-estradiol production by the corpus luteum is due to an increase in the expression of P450arom. In cattle, the pattern of P450arom expression and, hence, 17ß-estradiol synthesis differs dramatically from that in the human and rat. Similar to these species, estrogens are synthesized by granulosa cells of the follicle in cattle (18, 19); however, after ovulation, there is a rapid and permanent decline in both the synthesis of 17ß-estradiol and the expression of P450arom (20). Thus, even though both humans and cattle produce estrogens in their ovaries and placentas, the pattern of estrogen production differs between these two species. Therefore, regulation of transcription may also be distinct between the two species.

The loss of expression of P450arom with the onset of luteinization in the bovine, but not human, granulosa cell can also be duplicated in vitro. This occurs within 24 h of placing bovine granulosa cells in culture (20, 21). Consequently, due to the lack of a cell culture system in which aromatase activity can be maintained, much less is known about the regulation of expression of P450arom in the bovine ovary. We wished, therefore, to compare the regulation of expression of the ovary-specific CYP19 gene in these two species, namely the human CYP19 gene (hCYP19_ov) and the bovine CYP19 gene (bCYP19_ov). In this way we might determine whether cAMP drives transcription of the CYP19_ov gene in cattle in a fashion similar to that of the human CYP19_ov gene and thus gain greater insight into the regulatory mechanisms involved in ovarian steroidogenesis in cattle.

	Materials and Methods

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Restriction endonucleases were purchased from either Boehringer Mannheim (Indianapolis, IN) or Promega (Madison, WI) and used according to the directions supplied by the manufacturer. Forskolin was purchased from Calbiochem (San Diego, CA). Radiolabeled nucleoside triphosphates (3000 Ci/mmol) were purchased from DuPont-New England Nuclear Corp. (Boston, MA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.

Reporter gene constructs
Reporter gene constructs were prepared by creating 5'-deletion mutations of the human ovary-specific 5'-flanking region (promoter II) of the CYP19 gene as described previously (22). Preparation of the deletion constructs of the bovine ovary-specific 5'-flanking region followed the same procedure. Briefly, single stranded oligonucleotides were synthesized with sequences complementary to the bovine ovary-specific sequence of interest, with extra nonannealing ends for the restriction sites SalI (5'-primer) and PstI (3'-primer; Fig. 1). PCR products were subcloned into the pCRII vector using the TA Cloning System (Invitrogen, San Diego, CA). The PCR products were sequenced using Sequenase (version 1.0, U.S. Biochemical Corp., Cleveland, OH) to ensure the fidelity of the amplified sequences. The various deletion fragments were then subcloned into the SalI and PstI sites of a modified pGL3-Basic vector (pGL3-B_mod). Mutagenesis of the bovine cAMP response element (CRE)-like sequence (CLS) region to the hCLS was accomplished using PCR with an oligonucleotide containing the hCLS (italicized), some flanking sequence, and a nonannealing SalI site (5'-GAAtgCAcGTCACTCTACCCAC-3'; mutated nucleotides are shown in lowercase). To compare the human vs. bovine ovary-specific 5'-flanking regions, the Pileup Program from Genetics Computer Group, Inc. (GCG) (Madison, WI) was used. This comparison is shown in Fig. 1. As all bovine constructs were based on alignment with the human gene, the numbers corresponding to the location in the 5'-flanking region are not the same between the two species, even though they may span the same regions and encompass the same cis-acting elements.

View larger version (72K): [in this window] [in a new window]   Figure 1. Comparison of untranslated exons and 5'-flanking DNA for ovary-specific region of aromatase P450 in human (H) vs. cow (B). Asterisks in B sequences represent identity with the human nucleotide sequence. Dots represent gaps inserted by the Pileup program. There is 77% nucleotide identity between the H and B sequences. The forward- and backward-facing arrows represent the boundaries for the various H and B CYP19ov deletion constructs used for the transfection studies. The upstream sequence in boldface is that of the hCLS (-208/-201 bp), and the sequence in boldface and italics is the SF-1/Ad4BP binding sequence (-132/-125 bp). The downstream sequence in boldface is the TATA box (-31/-25), and the sequence just upstream from this site in boldface and underlined is the second TATA box in the bovine CYP19ov gene (12).

Transient transfection of both cell types was performed by electroporation as described previously (25) using 75 µg of the particular reporter construct plus 2 µg of the pCMVnlac ß-galactosidase expression plasmid as a control for transfection efficiency. For every transfection, a positive control vector (pGL3-control, Promega) as well as the empty vector (pGL3-B_mod) as a negative control were used. After transfection, cells were plated into 35-mm tissue culture dishes, such that there were six wells per construct. After recovery overnight in serum-containing medium, cells were starved in serum-free medium containing 0.1% BSA for 12 h, followed by treatment with either vehicle (n = 3/construct) or 25 µM forskolin (n = 3/construct) in serum-free medium plus 0.1% BSA for an additional 12 h.

Luciferase and ß-galactosidase assays
The luciferase assay was performed according to the manufacturer’s protocol using the Enhanced Luciferase Assay kit (Analytical Luminescence Laboratory, Ann Arbor, MI). ß-Galactosidase activity was measured using the Galacto-Light kit (Tropix, Bedford, MA), also following the protocol provided by the manufacturer. Luciferase and ß-galactosidase activities were assayed using a Monolight 2010 Luminometer (Analytical Luminescence Laboratory). Transfection efficiency was assessed by measuring the activity of ß-galactosidase expressed from the cotransfected ß-galactosidase expression vector. Relative luciferase activity was calculated by dividing the total luciferase activity per dish by the total ß-galactosidase activity per dish. All values shown for the various constructs have the relative activity of the empty vector (negative control) subtracted.

Electrophoretic mobility shift assays
Nuclear extracts were prepared from cultured bovine granulosa cells by the method of Dignam et al. (26). Protein concentrations were determined by a modified Bradford assay (Bio-Rad Laboratories, Richmond, CA). A double stranded oligonucleotide corresponding to the hCLS motif and flanking region (-217 to -198 bp; Figure 1) flanked by a 5'-MluI cohesive end and a 3'-SalI cohesive end was labeled by Klenow labeling using [-³²P]deoxy-CTP. A double stranded oligonucleotide corresponding to the analogous region of the bCYP19 gene (-212 to -194 bp; Fig. 1) was also labeled by Klenow fill-in. Nuclear protein (7.5 µg) was incubated with approximately 10,000 cpm probe as described previously (27). For the competition assays, various concentrations of nonradiolabeled hCLS or bCLS double stranded oligonucleotide were added simultaneously with the labeled probe. The resulting DNA-protein complexes were analyzed using 6% polyacrylamide gels with 0.5 x TBE electrophoresis buffer. Gels were dried and exposed to x-ray film.

	Results

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Expression of human and bovine CYP19_ov/luciferase reporter gene constructs in ovarian cells
To compare the cAMP-dependent regulation of the human vs. bovine CYP19_ov promoters, we created a series of nested 5'-deletion constructs of the bCYP19_ov to correspond to the hCYP19_ov gene, based on an alignment of the two promoter regions. These constructs are shown in Fig. 1. It should be noted that there is a high degree of sequence identity between the human and bovine CYP19_ov genes, approximately 70% up to -1000 bp and even greater than this closer to the start site of transcription (12). These fragments of the CYP19 promoter were ligated upstream of a luciferase reporter gene in the promoterless and enhancerless pGL3-B_mod vector. All of the constructs contained the endogenous human or bovine CYP19 TATA box. In the case of the bCYP19_ov gene, this means that two endogenous TATA boxes were present. The results of our previous study using the rapid amplification of complementary DNA ends (RACE) technique on RNA from bovine granulosa cells (12) showed that both TATA boxes drive transcription of the gene, although the one that aligns with the human CYP19_ov TATA box (i.e. the downstream one) appears to be the one primarily used. These reporter constructs were transfected into primary cultures of either bovine granulosa (in vitro luteinized) or luteal cells (in vivo luteinized). The transfected cells were treated for 12 h with 25 µM forskolin or vehicle. Cell lysates were assayed for both luciferase and ß-galactosidase activities. Results were similar whether the reporter constructs were transfected into bovine granulosa cells or bovine luteal cells; however, luciferase activity was usually lower in transfected luteal cells.

There was low basal expression of luciferase activity with all of the CYP19_ov constructs in the absence of forskolin (Fig. 2). As observed previously, it was not until the CLS sequence was included in the hCYP19_ov deletion constructs (-214/-16 bp) that luciferase activity could be induced with the addition of forskolin, even though one of the shorter constructs contained the SF-1 site (-140/-16 bp) (27, 28). The amount of luciferase activity stimulated by forskolin treatment could be increased further with the addition of more of the 5'-flanking sequence (-516/-16 and -693/-16 bp). The results obtained with the hCYP19_ov reporter constructs are in sharp contrast to those obtained with transient transfection of the bCYP19_ov constructs. There was little if any induction of luciferase activity with the addition of forskolin to the cells after transfection with any of the bCYP19_ov reporter constructs (-668/-12 to -96/-12 bp). Considering that there is no endogenous expression of P450arom in luteinized bovine granulosa cells in culture, these results did not appear too surprising. Upon closer inspection, however, it was noted that the sequence shown to be important in conferring the ability to respond to cAMP in the hCYP19_ov gene was not the same in the bCYP19_ov gene (ACGTCACT, hCLS vs. A¹GTCACT, bCLS) (12). To determine, therefore, whether this region of the bCYP19_ov gene is responsible for the lack of cAMP-stimulated bCYP19 gene reporter activity in luteinized cells, the bCLS (italicized) and flanking sequence were mutated so that they contained the hCLS (italicized) and flanking sequence in the context of the -209/-12 bCYP19_ov reporter construct [-209 M/-12 bp; GAACTCA¹GTCACTCTACCCAC (bovine) to GAAtgCAcGTCACTCTACCCAC (human)].

View larger version (18K): [in this window] [in a new window]   Figure 2. CYP19ov reporter gene construct expression in bovine granulosa cells in primary culture. Bovine granulosa cells were transfected with pGL3-Bmod containing -693/, -516/, -214/, -140/, or -100/-16 bp of 5'-flanking DNA of the hCYP19ov gene or -668/, -496/, -209/, -136/, or -96/-12 bp of 5'-flanking region of the bCYP19ov gene. A pGL3-control vector and the reporter plasmid with no insert were used as positive and negative controls, respectively. A ß-galactosidase expression vector was used to estimate transfection efficiency. Cells were treated for 12 h with either vehicle (ethanol; C) or forskolin (25 µM; F). Cell lysates were prepared and assayed for luciferase activity and ß-galactosidase activity as described in Materials and Methods. Data are expressed as luciferase activity relative to ß-galactosidase activity. This value for the negative control was subtracted from all values obtained for the constructs. This experiment was repeated 10 times. One representative experiment is shown. A similar pattern of relative luciferase activity, but at overall lower levels, was obtained when bovine luteal cells were transfected with these same constructs.

View larger version (17K): [in this window] [in a new window]   Figure 3. Mutation of the bCLS to the hCLS element causes the bovine -209/-12 bp construct (-209 M/-12 bp) to acquire cAMP responsiveness. Primary cultures of bovine granulosa cells were transfected with the human -214/-16 bp construct, the bovine -209/-12 bp construct, and the bovine -209/-12 bp construct with a mutation of the bovine to hCLS element (-209 M/-12 bp: see Materials and Methods for further details). Cells were transfected and treated as described in Fig. 2. Luciferase and ß-galactosidase activities were measured as described in Fig. 2. This experiment was repeated 10 times. A representative experiment is shown. Similar results were obtained, but at lower activities, when bovine luteal cells were transfected with these constructs.

View larger version (53K): [in this window] [in a new window]   Figure 4. EMSA shows granulosa cell nuclear protein binding to the hCLS (A), but very weak binding to the bCLS (B). Bovine granulosa cell nuclear protein (7.5 µg) was incubated with one of two 32P-labeled probes, either the hCLS (-217/-198 bp; A) or the bCLS (-212/-194 bp; B), in the absence (-) or presence of increasing amounts of cold hCLS or bCLS nonradiolabeled competitor (100-, 500-, or 1000-fold excess, indicated by the crescendo triangle). DNA-protein complexes were separated from free probe by electrophoresis and were visualized by autoradiography. fp, Free probe. The positions of the three specific complexes are indicated by A, B, and C.

Human CLS-nuclear protein complex A formation has been shown to be the result of CRE-binding protein homodimer interaction with this element. Most importantly, mutations within the hCLS that abolish complex A formation in vitro also abolish cAMP-induced reporter gene expression in bovine ovarian cells (22). As complex A formation does not occur in the presence of the radiolabeled bCLS, we propose that CRE-binding protein homodimer binding to this element does not occur, thus explaining at least in part the lack of cAMP-responsive gene transcription from the bovine CYP19_ov promoter.

	Discussion

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Aromatase is expressed in the ovary of all vertebrate species studied to date If the ovary-specific 5'-flanking regions and promoters from the human, bovine, rat, and chicken are aligned (12), there is a high degree of sequence identity among the genomic sequences of all four species. The SF-1/Ad4BP binding sequence (hCYP19_ov, -132/-125 bp), which has been shown to be essential for the expression of a number of steroidogenic enzymes (29, 30), and the TATA box (-31/-25 bp) sequence are perfectly conserved in all four species. Thus, there is a high degree of conservation not only of general sequence among the various species, but of specific cis-acting elements as well. These results might be expected due to the similarities in estradiol synthesis of the growing follicle among the various mammalian species; however, the bovine corpus luteum does not synthesize estradiol, whereas that of the human does. We wished to compare, therefore, the regulation of the bovine vs. the human CYP19_ov promoters and 5'-flanking DNA. Deletion constructs of the bCYP19_ov gene were made that corresponded to hCYP19_ov reporter constructs used in previous experiments conducted in our laboratory (22, 27). Both the human and bovine CYP19_ov reporter constructs were transfected into bovine ovarian cells. We observed the expected pattern of reporter gene activity from cells transfected with the hCYP19_ov reporter constructs (Fig. 2); in constructs that did not include the CLS (-100/-16 and -140/-16 bp), there was no increase in reporter gene activity upon the addition of forskolin. With the addition of the CLS (-214/-16 bp and larger), there was an increase in reporter gene activity upon the addition of forskolin, however, the results were quite different when the bCYP19_ov deletion constructs were employed. In no instance did the addition of forskolin to the transfected cells increase reporter gene activity over the values obtained for the empty vector. These results were not surprising, as the endogenous bCYP19 gene is not expressed in luteinized bovine granulosa cells or luteal cells (20, 21, 31).

We thought that the difference between the expression of the human vs. bovine CYP19_ov reporter constructs was simply a case of the presence of a cis-acting element(s) in the hCYP19_ov gene that promoted activation or, conversely, the presence of a cis-acting element(s) in the bCYP19_ov gene that repressed activation even when trans-acting factors were present with the onset of luteinization. As both the human and bovine CYP19_ov constructs were transfected into the same cell type at the same time, the differences could not simply be the result of different trans-acting factors being present in a particular cell type. Upon closer inspection of the bCYP19_ov sequence, it was noted that there was a one-nucleotide deletion in the CLS, which, as stated earlier, was shown to be essential in both the human (22) and the rat (32) CYP19_ov gene to permit induction of reporter constructs in ovarian cells by cAMP. We, therefore, mutated the bCLS to the hCLS, to determine whether this might explain the lack of an increase in reporter gene activity of the bCYP19_ov constructs with the addition of forskolin. The mutation of the bovine to hCLS and flanking sequence caused an increase in reporter gene activity with the addition of forskolin (-209 M/-12 bp); however, this increase was never equal to that of the wild-type human construct (-214/-16 bp). These results are further corroborated by the results of the EMSAs. When the hCLS was used as a radiolabeled probe, unlabeled bCLS could not displace the hCLS-protein complex formed with granulosa cell extract. When the bCLS was used as a radiolabeled probe, similar bCLS-protein complexes were formed; however, the intensity of the interaction was extremely weak compared with that of the hCLS. These results were obtained when probes radiolabeled at similar efficiencies were employed and the blots were exposed to film for the same length of time. To determine whether the deletion in the bCLS might simply be an artifact of the genomic library used to obtain the bCYP19_ov clone, we amplified the genomic region containing the CLS from bovine genomic DNA obtained from adrenal tissue procured from a local slaughterhouse. The results were the same; namely, that the bovine genome appears to have a deletion at this site (data not shown). It, therefore, appears as if through evolution, the cow has lost a cis-acting element important for cAMP-induced transcription of the CYP19_ov gene in other species.

This species-specific difference in the presence of CREs in a given gene is not unique to the CYP19 gene. The glycoprotein hormone -subunit gene is expressed in the pituitaries of all mammals, but its expression in placenta is limited to only primates and horses. The 5'-flanking region of the human -subunit gene contains tandem palindromic repeats of CREs (33). Conversely, in a number of species that do not express the glycoprotein hormone -subunit gene in their placentas, namely cattle and rodents, there is a single CRE in the 5'-flanking region of the -subunit gene that differs from the human gene by one nucleotide. Expression of the human -subunit gene is enhanced by the addition of cAMP in a human choriocarcinoma cell line (BeWo). The bovine -subunit gene is not expressed in these cells; however, with mutation of the bovine to the human CRE, the bovine -gene does gain both cAMP responsiveness as well as placenta-specific expression (33). Another example of species-specific expression of genes is the case of the plasminogen activators (PA). In the rat granulosa cell, tissue-type PA (tPA) is induced by gonadotropins through a cAMP-dependent pathway, which correlates with the time of ovulation of the follicle in vivo. In mice and humans, however, rather than tPA being expressed at the time of ovulation, a related, but distinct, protease is induced, namely urokinase PA. Comparison of the 5'-flanking regions of the tPA gene in these three species revealed a consensus CRE in the rat tPA gene, but a one-nucleotide substitution in the mouse and human tPA gene (34). Conversion of the mouse and human CRE to the rat CRE caused the promoters from these two to respond with increased activity after treatment with cAMP. Conversely, mutation of the rat CRE to the analogous mouse and human CRE abolished cAMP responsiveness of the rat tPA promoter. Thus, this is an example of two different enzymes being expressed in different species to accomplish the same physiological function.

From both in vivo (19) and in vitro (18) studies in the cow, it is known that granulosa cells of the preovulatory follicle express P450arom and produce 17ß-estradiol (20); however, circulating levels of 17ß-estradiol are much lower in the cow than in women (16, 17, 35). When bovine granulosa cells are cultured in vitro, aromatase activity drops within 12 h (20, 21). Therefore, experiments to study the expression of bovine P450arom in vitro have been difficult. There are some reports of cultures of bovine granulosa cells that maintain very low activity in vitro (21, 36). The lack of a consensus CLS in the bovine sequence may be the explanation for these findings. There, of course, is the possibility that the bovine gene has recruited another as yet unidentified sequence to serve as a CRE, either further downstream from -12 bp or further upstream from -668 bp. It is intriguing that the bovine species may have developed a mechanism for regulating transcription of the CYP19_ov gene that is very different from that in the human or the rat, even with such a high degree of conservation of sequence among the three species.

Even when the bCLS and its flanking sequence were changed to those of the hCLS and flanking sequence (-209 M/-12 bp), the increase in reporter gene activity did not match that in the wild-type human construct (-214/-16 bp). Consequently, only a part of the lack of response to forskolin by the bCYP19_ov constructs can be explained by the lack of a CLS. These results are further corroborated by the studies of Michael and Simpson (22), in which dinucleotide mutations were performed on the region from -142 to -125 bp (GGGAATGCACGTCACTCT). These mutagenized sequences were used as nonradiolabeled competitors for EMSA studies employing the wild-type CLS region as a probe, and/or they were subcloned into the pGL3-B_mod vector, just 5' of the -140/-16 bp hCYP19_ov construct. When AC (shown in italics above) was mutated, it was not able to compete for nuclear protein binding of complexes A and B to the wild-type hCLS. When this mutation was included upstream of the -140/-16 bp hCYP19_ov construct and transfected into bovine luteal cells, luciferase activity was only 15–20% that of the wild-type sequence ligated upstream of the -140/-16 bp hCYP19_ov construct. The only other sequences that differ between the hCLS and bCLS regions are the TG (shown in boldface above). When these two nucleotides were mutated, this oligonucleotide remained effective in competing for nuclear protein complexes A and B bound to the wild-type hCLS probe. It appears that the TG dinucleotide may not be important in the cAMP response of the hCYP19_ov gene. Based on our results and those of Michael and Simpson (22), we conclude that there must be some other sequence(s) in this region of the bovine gene preventing expression of the bCYP19_ov, perhaps due to the presence of a repressive trans-acting factor expressed with the onset of luteinization of the bovine granulosa cell. Conversely, some sequence(s) may be present in this region of the hCYP19_ov gene that is important in the induction of transcription by cAMP but has yet to be identified and is not present in the bCYP19_ov gene.

	Footnotes

 
¹ This work was supported in part by USPHS Grant HD-13243 and in part by USPHS Training Grant GM-08203 (to M.D.M.).

Received November 15, 1996.

	References

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