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Endocrinology Vol. 143, No. 10 4031-4037
Copyright © 2002 by The Endocrine Society

ARTICLE

A Polymorphic Form of Steroidogenic Factor-1 Is Associated with Adrenocorticotropin Resistance in Y1 Mouse Adrenocortical Tumor Cell Mutants

Claudia Frigeri, Jennivine Tsao, Martha Cordova and Bernard P. Schimmer

Banting and Best Department of Medical Research (J.T., M.C., B.P.S.) and Department of Pharmacology (C.F., B.P.S.), University of Toronto, Toronto, Ontario, Canada M5G 1L6

Address all correspondence and requests for reprints to: Bernard P. Schimmer, Ph.D., Professor, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada. E-mail: bernard.schimmer{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ACTH resistance in mutant derivatives of the Y1 mouse adrenocortical tumor cell line results from a defect that affects the activity of steroidogenic factor-1 (SF1), thereby preventing the expression of the melanocortin-2 receptor. In this report, we show that the SF1 genes in ACTH-resistant mutants differ from the gene in ACTH-responsive Y1 cells by two base changes—one that changes an Ala to Ser at codon 172, and one in the third position of codon 3 that does not affect the protein sequence. Furthermore, several of the mutants contain multiple copies of this alternate SF1 gene (SF1S172) on acentric chromosome fragments. The SF1S172 allele represents a polymorphism rather than a spontaneous mutation because the two SF1 alleles can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1A172 allele also is found in C57Bl/6J and C57Bl/10J mice, whereas the SF1S172 allele also is found in C3H/HeJ and DBA/2J mice. The two forms of SF1 had only modest differences in activity suggesting that the SF1 polymorphism per se is not directly responsible for ACTH resistance. Our results indicate that the SF1S172 allele is a marker of ACTH resistance in this family of adrenocortical tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROIDOGENIC FACTOR 1 (SF1) is a member of the nuclear receptor family of transcription factors that has a global role in endocrine function, regulating the development and activity of the hypothalamic-pituitary-gonadal axis and the development and function of the adrenal cortex (reviewed in Ref. 1). The importance of SF1 in endocrine development has been elegantly demonstrated in studies of SF1-disrupted mouse models (2, 3, 4, 5, 6) and in two human patients with SF1 mutations (7, 8). SF1 also is thought to be required for the expression of many of the genes required for steroid hormone biosynthesis in the adrenal gland and gonads—e.g. genes encoding the cytochrome P450 steroid hydroxylases, 3ß-hydroxysteroid dehydrogenase, steroidogenic acute regulator (StAR), and the ACTH receptor (mc2r) (1). The contributions of SF1 to the regulated expression of specific genes, however, have been inferred largely from analyses of the activities of proximal promoter sequences in transfection studies—an approach that may overestimate the importance of SF1 (9).

Previously, we isolated and characterized several mutants derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line that harbors defects that compromise SF1 function (10). The defects in these mutants did not affect the ability of SF1 to bind to specific recognition sites on DNA but did impair the activation functions of the protein (10, 11). These mutants exhibited a complex phenotype of ACTH resistance that included the impaired expression of a number of genes required for steroidogenesis, including the mc2r, Cyp11a, Cyp11b1, and StAR (10, 12, 13). Thus, these ACTH-resistant mutants provide a potentially useful system to evaluate the importance of SF1 in gene expression.

The present study was undertaken to determine whether differences in the SF1 gene are associated with altered SF1 function in the ACTH-resistant mutants. We report that the ACTH-resistant mutants have a SF1 allele that differs from the allele found in ACTH-responsive Y1 cells and that this allele is present in some of the mutants in multiple copies on acentric chromosome fragments. The SF1 allele found in the mutant clones represents a polymorphism rather than a spontaneous mutation and can be traced to the F1 mouse (C57L/J x A/HeJ) from which the original tumor was derived (14). Thus, the SF1S172 allele is a marker of ACTH resistance among cell populations derived from this mouse adrenal tumor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA, DNA, plasmids, and oligonucleotides
Total cellular RNA was isolated from Y1 and mutant cells using a RNA-easy Mini kit (QIAGEN Canada, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. SF1 cDNA was amplified from total RNA by RT-PCR using amplification primers derived from the genomic sequence of SF1 (15). First-strand cDNA synthesis was performed using oligo(deoxythymidine)18 as primer and Superscript II Reverse Transcriptase (Invitrogen Canada, Burlington, Ontario, Canada). SF1 cDNA was amplified by PCR using Taq DNA polymerase (2.5 U, Invitrogen Canada), and 50 pmol each of a sense oligonucleotide starting 31 bp upstream of the initiator ATG (5'-ATTCTCCTTCCGTTCAGCG-3') and an antisense oligonucleotide starting 183 nucleotides downstream of the translation stop codon (5'-CAGGCCAATGGCTAGTAAAGG-3') in a volume of 50 µl. PCR conditions were 94 C for 1 min, 62 C for 1 min, 72 C for 1 min over 32 cycles with a hot start. Products were either sequenced directly or sequenced following cloning into the vector pCR2.1 A/T (Promega Corp., Madison, WI). Sequences were determined using an automated DNA sequencing facility at The Hospital for Sick Children (Toronto, Ontario, Canada). SF1 expression vectors were prepared by subcloning SF1 from pCR2.1 into the EcoRI site of pcDNA3.1+ (Invitrogen Canada) downstream of a cytomegalovirus core promoter and enhancer. A 13-kb genomic SF1 fragment for hybridization analysis was prepared by PCR amplification of BAC clone 10I15 using primers derived from the SF1 genomic sequence (15) within the third exon (5'-GCATTACACGTGCACCGAG-3') and from a complementary sequence within the sixth exon (5'-CAGGCCAATGGCTAGTAAAGG-3'). PCRs were performed using the Expand Long Template PCR System (Roche Diagnostics, Laval, Québec, Canada) with a hot start. Samples were subjected to 10 cycles of incubation at 94 C for 10 sec, 62 C for 30 sec, and 68 C for 8 min and 20 cycles of incubation under the same conditions, except that period of incubation at 68 C was extended by 20 sec for each cycle. Samples then were incubated at 68 C for an additional 7 min to complete the reaction. High molecular weight genomic DNA from cells and from the livers of C57L/J and A/HeJ mice was isolated by phenol/chloroform extraction and ethanol precipitation as described in detail elsewhere (16). Livers from C57L/J and A/HeJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME); DNA samples from other mouse strains were purchased directly from The Jackson Laboratory.

Cell culture and gene transfer
Cells were routinely cultured as monolayers at 36.5 C under a humidified atmosphere of 95% air-5% CO2 in nutrient mixture F10 supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated fetal bovine serum, penicillin G, and streptomycin sulfate as described previously (17). Tissue culture reagents were obtained from Invitrogen Canada.

For gene transfer experiments, cells (2 x 105/dish) were replicate plated in 60-mm tissue culture dishes and grown for 3 d. Cells were transferred to {alpha}-MEM supplemented with serum and antibiotics and transfected with supercoiled plasmid DNA using a high-efficiency calcium phosphate precipitation technique (16). Cells were incubated with the DNA precipitates for 24 h, rinsed to remove the DNA and incubated in fresh medium for 48 h.

Luciferase activity
Cells were harvested by scraping into a lysis buffer (50 mM Tris.2-[N-morpholino]ethanesulfonic acid, pH 7.8; 1% Triton X-100; 4 mM EGTA; and 1 mM dithiothreitol), disrupted by vortexing, and clarified by centrifugation at 4 C. Cell supernatants (4–400 µg protein) were assayed for luciferase activity in a reaction cocktail (250 µl) containing 200 µl of cell extract; 45 mM Tris.2-[N-morpholino]ethanesulfonic acid, pH 7.8; 9 mM magnesium acetate; 3 mM disodium ATP; and 60 µM luciferin as described (18). Luminescence was measured using a Berthold Lumat LB luminometer under conditions where signals were proportional to the amount of cell extract added to the reaction.

Fluorescent in situ hybridization analysis (FISH) analysis
Chromosome spreads were prepared from Y1 and mutant cells following treatment with vinblastine (1 µg/ml) for 6 h (19), visualized by staining with the chromatin binding fluorescent dye 4'-6'diamidino-2-phenylindole (DAPI) and probed for SF1 and for a marker of the distal region of chromosome 2. The SF1 probe was prepared by nick-translation of the 13-kb SF1 genomic fragment in the presence of digoxigenin-11-deoxyuridine triphosphate and detected using a rhodamine-labeled rabbit anti-digoxigenin antibody. The marker for the distal region of mouse chromosome 2 was prepared by nick-translation of BAC clone 349o16 in the presence of biotin-14-dATP and detected using fluorescein isothiocyanate-labeled streptavidin. The localization of SF1 was determined by superimposing images of the hybridization signals for SF1 and chromosome 2 upon the images of the DAPI-stained chromosomes. Labeling and FISH reactions were carried out by the Center for Applied Genomics, Hospital for Sick Children.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence differences in the SF1 genes from parent and mutant cells
SF1 transcripts from Y1 cells and from the ACTH-resistant mutants 10r6, 10r9, OS3 and Y6 were amplified by RT-PCR and the resultant cDNAs were sequenced. The SF1 cDNAs from mutant 10r6, 10r9, and OS3 cells differed from that of Y1 at two positions—each mutant had a C instead of T at position 9 relative to the start of translation that did not affect the amino acid sequence and a T instead of G at position 514 that changed codon 172 from an Ala (GCT) to a Ser (TCT) and disrupted a unique NcoI site (Fig. 1Go). This difference in the SF1 transcripts between parent and mutant cells was confirmed by NcoI digestion of the PCR products (Fig. 2AGo). SF1 cDNA amplified from Y1 cells yielded two fragments (1060 bp and 540 bp) when treated with NcoI whereas SF1 cDNAs from the three mutants were largely resistant to digestion by the enzyme. Trace amounts of an NcoI-sensitive form of SF1 were evident in each mutant suggesting that the parental form of SF1 was present at low levels in these clones. In the mutant Y6 clone, the two forms of SF1 were more equally represented as determined by digestion with NcoI (Fig. 2AGo) and as confirmed by sequence analysis (not shown).



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  		Figure 1. Nucleotide sequences of SF1 from Y1 and mutant 10r6, 10r9 and OS3 cells. RNA was amplified from Y1 and mutant cells by RT-PCR and sequenced as described in Materials and Methods. The sequence of SF1 from parent Y1 cells (GenBank accession no. AF511594) is presented with the two base changes observed in the mutant clones noted below the Y1 sequence. Amplification primers used for the PCRs are underlined; the arrow indicates the start of translation; the asterisk indicates the stop codon, the box surrounds the unique NcoI site.

 


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  		Figure 2. Identification of NcoI-sensitive and NcoI-resistant SF1 alleles by restriction digestion. SF1 cDNA (panel A) was amplified from total RNA of Y1 and mutant cells as described in Materials and Methods. Exon 3 of the SF1 gene (panel B) was amplified from genomic DNA of Y1 and mutant cells and from the mouse strains A/HeJ and C57L/J by PCR using 5'-TGCGTGCTGATCGAATGC-3' and 5'-CCAGTCGACAATGGAGATAAAGG-3' as forward and reverse primers, respectively. PCR conditions were 94 C 1 min, 60 C 1 min, 72 C 1 min for 30 cycles followed by a 10-min incubation at 72 C. In panels A and B, products were digested with the restriction endonuclease NcoI, resolved by electrophoresis on agarose gels, and visualized by staining with ethidium bromide. Fragment sizes were estimated using a DNA ladder. In panel C, genomic DNA (10 µg) from Y1 and mutant cells was incubated in the absence (-) or presence (+) of NcoI and analyzed by Southern blot hybridization using a nick-translated cDNA probe containing the SF1 sequence from +168 to +740 (Fig. 1Go). Signals were visualized using a phosphorimager (Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada). The NcoI fragments obtained with DNA from Y1 cells are indicated (arrows).

 
The distribution of the two SF1 genes seen at the cDNA level reflected their genomic distribution. DNA fragments (593 bp) encoding the SF1 amino acids 84–280 and including the diagnostic NcoI site (15) were amplified from genomic DNA of Y1 and mutant cells and digested with NcoI to distinguish the two SF1 alleles (Fig. 2BGo). The PCR product of the SF1 gene from Y1 cells was completely digested by NcoI, yielding two fragments of approximately 331 bp and 262 bp indicative of the SF1A172 allele. The PCR products of amplified genomic fragments from the 10r6, 10r9, and OS3 mutants were largely resistant to digestion by NcoI, indicating the predominance of the SF1S172 allele; however, traces of NcoI-digested fragments also could be seen on the gels. The PCR fragment from the Y6 mutant contained both the NcoI- sensitive and NcoI–resistant forms in amounts that reflected the proportions of the two SF1 transcripts.

Amplification of SF1 in mutant cells
The proportions of the two SF1 alleles were examined by Southern blot hybridization (Fig. 2CGo). SF1 signals were much stronger in the undigested DNA samples from mutant 10r6, 10r9, and OS3 cells than in the corresponding samples from Y1 cells, despite the loading and blotting of equivalent amounts of total DNA. NcoI digestion revealed that the increased signal intensity for SF1 in three mutants was due primarily to the NcoI-resistant allele; the levels of the NcoI-sensitive SF1 allele were approximately equal to that in Y1 cells. Taken together, these results indicated that the NcoI-resistant SF1 allele was selectively amplified in these three mutant clones. The SF1 signals in the Y6 mutant approximated those seen in Y1 cells and the two SF1 alleles appeared to be in roughly equal proportions.

The nature of the SF1 amplification was further explored by FISH analysis of metaphase spreads prepared from Y1 and mutant cells. As shown in Fig. 3AGo, metaphase spreads of Y1 cells contained two copies of chromosome 2 as evidenced by the labeling of the distal ends with the reference marker for this chromosome (green). The SF1 probe (red) labeled the same two chromosomes near the centromere. These findings demonstrate that Y1 cells are diploid for the SF1 gene, which is known to reside on the proximal part of chromosome 2 (20, 21). The10r9 and OS3 mutants also are diploid for chromosome 2 as evidenced by the two signals for the chromosome marker in interphase nuclei and by the labeling of two chromosomes in metaphase spreads; however, these mutants contained multiple signals for the SF1 gene, most of which were extrachromosomal and not associated with the DAPI-stained structures (Fig. 3Go, B and C). The 10r6 mutant was polyploid with a median chromosome number of 68 that included three copies of chromosome 2; in addition, 10r6 contained multiple extrachromosomal signals for SF1 (data not shown). Thus these mutants contain multiple copies of SF1, with many on structures too small to visualize by standard staining techniques. In the mutant Y6, SF1 was localized to the two copies of chromosome 2 and, unlike the other mutants, did not contain SF1 on extrachromosomal fragments.



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  		Figure 3. FISH analysis of the SF1 gene in Y1 and mutant cells. Chromosome spreads were prepared, stained with DAPI (blue), and labeled with probes for the SF1 gene (red) or the distal end of chromosome 2 (green) as described in Materials and Methods. Panels A and D show metaphase spreads from Y1 and Y6 cells respectively; panels B and C show interphase nuclei and metaphase spreads from mutant 10r9 and OS3 cells, respectively. The arrows identify the chromosome 2 pairs in the metaphase spreads.

 
To determine whether the gene amplification impacted on the levels of SF1 protein in the mutant clones, whole-cell extracts were compared for SF1 expression on Western blots. We had reported previously that SF1 levels were not diminished in the mutant clones 10r6 and 10r9 (10). In fact, as shown in Fig. 4Go, SF1 protein levels were higher in clones10r6, 10r9 and OS3 than in Y1 cells whereas the level of SF1 in the Y6 mutant approximated that in the Y1 cell line. These results thus indicate that the amplification of the SF1 gene in mutant clones resulted in higher levels of SF1 protein.



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  		Figure 4. Western blot analysis of SF1 in parent and mutant cells. Whole-cell extracts from parent and mutant cells (25 µg protein) were electrophoresed on 10% polyacrylamide gels in the presence of SDS, blotted onto nitrocellulose, and probed for SF1 using a rabbit anti-SF1/AD4BP antibody generously provided by K. Morohashi (National Institute of Basic Biology, Okazaki, Japan). Antigen-antibody interactions were detected by chemiluminescence using a horseradish peroxidase-labeled secondary antibody and the Renaissance chemiluminescence reagent (NEN Life Science Products, Boston, MA).

 
Transcriptional activity of SF1A172 and SF1S172
The transcriptional activities of SF1A172 and SF1S172 were compared in transient transfection assays using a SF1- dependent reporter gene assay. Y1 cells were transfected with expression vectors encoding SF1A172 or SF1S172 (0.5 µg) together with a luciferase reporter plasmid (1.0 µg) driven by three copies of the SF1 site at position -25 of the mc2r gene (10) or with a luciferase reporter plasmid (2.0 µg) driven by five copies of the SF1 site at -65 in the cyp21 gene (22). SF1S172 had approximately half (46 ± 5%, n = 4, P < 0.05) the activity of SF1A172 when assayed using the mc2r-derived SF1 reporter construct; however, the activities of the two SF1 constructs were indistinguishable in the context of the Cyp21-derived SF1 reporter vector (data not shown). The transcriptional activities of the two SF1 alleles were similar in other cell contexts as well. SF1S172 had 94 ± 4%, n = 3 and 117 ± 20%, n = 3 of the activity of SF1A172, respectively, in steroid- secreting mouse I-10 cells (23) and in COS-1 African green monkey cells that do not have endogenous SF1 (24).

Distribution of SF1A172 and SF1S172 among mouse strains
We next considered the possibility that the SF1S172 gene seen in the ACTH-resistant mutant clones represented a naturally occurring polymorphism that segregated with ACTH resistance rather than a mutation that arose de novo. Inasmuch as the Y1 cell line and derivative mutants all were derived from a tumor originating in an offspring of a C57L/J female mated with an A/HeJ male (14), these two mouse strains were examined for polymorphisms in the SF1 gene. The SF1 gene region containing the diagnostic NcoI site, when amplified from the genomic DNA of the C57L/J strain, yielded 331-bp and 262-bp products following NcoI digestion indicating the presence of the SF1A172 allele; the corresponding region of the SF1 gene from the A/HeJ strain yielded only the original 593 bp fragment following NcoI digestion indicating the presence of the SF1S172 allele (Fig. 2BGo). Analysis of the DNA sequences of these PCR products and of PCR products corresponding to the region surrounding codon 3 confirmed that the C57L/J strain contained the SF1A172 allele which included the G in the first position of codon 172 and the T in the third position of the third codon whereas the A/HeJ strain contained the SF1S172 allele, which included the T in the first position of codon 172 and the C in the third position of the third codon (data not shown). These findings thus indicate that the two different forms of SF1 seen in Y1 and mutant cells do not represent spontaneous mutations; rather, they represent polymorphisms originating in the parental mouse strains from which the cells were derived.

We also examined the distribution of SF1 alleles among four other mouse strains. As shown in Fig. 5Go, the NcoI- sensitive SF1A172 allele also is found in strains C57Bl/6J and C57Bl/10J carried while the NcoI-resistant SF1S172 allele is associated with strains C3H/HeJ and DBA/2J.



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  		Figure 5. Distribution of SF1 alleles among mouse strains. Exon 3 of the SF1 gene (593 bp) was amplified from genomic DNA of mouse strains C3H/HeJ, C57Bl/10J, C57Bl/6J, and DBA/2J as described in the legend to Fig. 1Go. PCR products were digested with the restriction endonuclease NcoI, resolved by electrophoresis on agarose gels, and visualized by staining with ethidium bromide. Fragment sizes were estimated using a DNA ladder. The generation of two fragments (331 bp and 293 bp) following NcoI digestion is indicative of the NcoI-sensitive SF1A17s allele. The NcoI-resistant 593-bp fragment is indicative of the SF1S172 allele.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The origins of the four mutants described in this report are outlined in Fig. 6Go. The original ACTH-responsive Y1 cell line and the ACTH-resistant clone Y6 were isolated by random cloning from a transplantable adrenocortical tumor (25, 26). The ACTH-resistant mutant, OS3, arose spontaneously from the Y1 cell line (27). Y1BS1, the ACTH-responsive cell line used in this study, was isolated as a phenotypically stable subclone of the original Y1 population (14). The ACTH-resistant clones, 10r6 and 10r9, were isolated from Y1BS1 cells by selective growth in the presence of the diterpene forskolin (28). Despite their apparently independent origins, the four mutants have complex phenotypes that are remarkably similar. All four clones owe their ACTH resistance to a defect that impairs SF1 function (Refs. 10 and 11 ; and our unpublished observations), thereby inhibiting expression of the mc2r and other SF1-dependent genes (12, 13). The mutants also exhibit secondary deficiencies in Gs{alpha} and Gi{alpha} that are caused by the loss of the mc2r and that can be restored by transfecting the mutants with a mc2r gene under the control of the cytomegalovirus promoter and enhancer (13). In addition, all the mutants are resistant to the growth inhibitory and morphological effects of forskolin (13, 28). Our finding that the ACTH-responsive Y1BS1 clone has the SF1A172 allele, whereas the four mutant clones all have the SF1S172 allele suggests that the SF1S172 allele is a marker of the mutant phenotype in this mouse adrenocortical tumor cell system.



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  		Figure 6. Origins of Y1, Y6, OS3, 10r6, and 10r9 clones. The original ACTH-responsive Y1 clone and the ACTH-insensitive variant Y6 (25 26 ) were isolated from a transplantable mouse adrenal cortical tumor that arose following atomic irradiation of the F1 cross between a C57L female and a A/HeJ male mouse (33 ). ACTH-resistant OS3 cells arose spontaneously from the parent Y1 clone. Clone Y1BS1 is an ACTH-responsive subclone of the original Y1 population that was randomly isolated following single-cell plating and screening for sensitivity to ACTH (14 ). Clones 10r6 and 10r9 were isolated from Y1BS1 as colonies resistant to the growth inhibitory and morphological effects of 10 µM forskolin (28 ).

 
In clones 10r6, 10r9, and OS3 (Fig. 3Go and our unpublished observations), the SF1S172 allele is amplified on acentric chromosome fragments. Acentric chromosome fragments are commonly found in tumor cells and are thought to arise from chromosome breakage, followed by asymmetric segregation of the chromosome and its fragments during mitosis. The fragments often contain several genes from the originating chromosome, and sometimes include genes that confer a selective growth advantage, favoring their retention during mitosis (29). The asymmetric segregation of chromosome 2 probably accounts for the observation that the Y1BS1 clone is homozygous for the SF1A172 allele and not heterozygous for the two SF1 alleles as might have been predicted from the genotype of the F1 mouse from which the adrenal tumor arose. Clearly, a very small fraction of the Y1BS1 population must still contain the SF1S172 allele on acentric fragments because mutants such as 10r6 and 10r9 are present at low frequency in the Y1BS1 population (28). We also have found that a large fraction of the parent Y1 cell line (Fig. 6Go), which we obtained from the American Type Culture Collection (Manassas, VA), contains the amplified SF1S172 allele and is resistant both to ACTH and forskolin (our unpublished observations). Thus, the acentric fragments containing SF1S172 alleles can be traced back to the original Y1 clone and likely arose in the tumor either during its formation or during its subsequent passage from mouse to mouse. Interestingly, of the clones examined only theY6 clone is clearly heterozygous for the two SF1 alleles. Because we cannot readily distinguish the chromosomal alleles of SF1 in the presence of the amplified acentric fragments, we cannot determine which SF1 alleles are present on the chromosome 2 pairs in the other mutant clones.

The association of SF1S172 with ACTH resistance raises the possibility of a linkage between the SF1 genotype and the mutant phenotype. Because the activity of SF1S172 was at most only modestly different from that of SF1A172 in SF1-dependent reporter gene assays, the SF1S172 allele per se likely is not responsible for the severely impaired expression of the mc2r or for the markedly reduced SF1 activity seen in the mutant clones (10, 12, 13). Inasmuch as the mutations in clones 10r6, 10r9 and OS3 are associated with amplification of the SF1 gene on acentric fragments, ACTH resistance in these clones may have resulted from an excess of SF1, which in turn sequestered essential transcription factors and cofactors away from SF-1-dependent promoters (a process termed "squelching"; Ref. 30); however, although the Y6 mutant shares many features of the ACTH-resistant phenotype with the other ACTH-resistant mutants, the SF1S172 allele does not appear to be appreciably amplified in this clone, arguing against SF1 amplification as an underlying cause. It is thus possible that another gene closely linked to the SF1S172 allele is responsible for the mutant phenotype. This putative gene might act by repressing SF1 function either through a direct or indirect mechanism.

The deduced SF1 protein sequences from mouse (AAB28338), rat (A56120), pig (P79387), cow (Q04752), horse (AAG35648), and human (NP 004950) genes deposited in GenBank are 89% identical and all have Ala at residue 172. Although an SF1 with Ser at residue 172 had not been described previously, elp-1 (NM 139050) and elp-3 (NM 0080), alternate splice products of the SF1 gene from the 129 mouse strain, have Ser at the position corresponding to residue 172 in SF1 and a C instead of T in the third position of the codon corresponding to codon-3. These observations suggest that the 129 mouse strain, like strains C3H/HeJ and DBA/2J, contains the SF1S172 allele. While SF1 alleles in mouse Y1 cells and in the Balb C mouse (15), both encode a transcript with Ala at residue 172, the Balb C mouse contains a C instead of T in the third position of codon three and is missing 3 of the 5 bases in the 3'-untranslated region from nucleotide 1457 (Fig. 1Go). These differences do not impact on the protein product; however, they may denote an additional allelic variant of SF1. Interestingly, testicular Leydig cells from the mouse strains C57Bl/10J and C57Bl/6J that contain the SF1A172 allele (Fig. 5Go) have been reported to produce approximately twice the amount of testosterone in response to human CG stimulation than cells from the mouse strains C3H/HeJ and DBA/2J carrying the SF1S172 allele (31, 32). Although the genetic basis underlying this difference in steroidogenic potential among mouse strains has not been defined, it is tempting to speculate that the two SF1 alleles may serve as markers of steroidogenic potential among mouse strains as well.


   Acknowledgments
 
We gratefully acknowledge Jo-anne Herbrick, Martin Li, and Lap-chi Tsui for assistance with the FISH analyses and for provision of the BAC clones used in this study; Natasha Kraeva and David MacLennan for assistance with DNA sequence analyses; Jeff Milbrandt for the Cyp21-derived SF1 reporter gene; Ken Morohashi for the SF1 (Ad4BP) antiserum; and Keith Parker for critical reading of the manuscript and helpful discussions.


   Footnotes
 
This work was supported by research grants (to B.P.S.) and a studentship award (to C.F.) from the Canadian Institutes of Health Research. The Y1 cell line used in this study is Y1BS1 (14 ), a stable subclone of the cell line originally isolated by Yasumura et al. (25 ).

Abbreviations: DAPI, 4'-6'Diamidino-2-phenylindole; FISH, fluorescent in situ hybridization analysis; mc2r, ACTH receptor; SF1, steroidogenic factor-1 (NR5A1); StAR, steroidogenic acute regulator.

Received March 26, 2002.

Accepted for publication June 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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