We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a A Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another A clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.The rat seminal vesicle, under the influence of testosterone, produces a group of small basic polypeptides referred to as seminal vesicle secretion IV and V (SVS IV and V) (1, 2). We have isolated an llS poly(A)+ mRNA fraction that codes for precursors to SVS IV and V (3). The complementary DNA (cDNA) to the mRNA was cloned in Escherichia coli by using plasmid pBR322. Clones were identified and cDNAs were sequenced that contain DNA information for SVS IV and V mRNA (4, 5). It was shown, by using SVS IV cDNA as probe, that the mRNA for SVS IV is rapidly induced after testosterone administration to rats castrated for several weeks (4, 6).One clone, pSV2, has a cDNA insert complementary to SVS IV mRNA and 540 base pairs (bp) long (4). The cDNA insert from pSV2 was used as a probe to screen a rat gene library prepared in phage A Charon 4A. Several positive phage signals were identified and the rat DNA constituting the genomic DNA sequences for the SVS IV gene was then characterized by restriction mapping and DNA sequence analysis. To our surprise, the SVS IV gene exists in two forms, which differ by the number of 20-bp tandem repeats in an intron. This intron size variability in the SVS IV gene was shown to be an allelic variant by the appropriate genetic crosses. A major repetitive element was also identified just 3' to the end of the transcription unit. 32P-Labeled DNA probes were prepared by nick-translation (4). To identify genomic clones of the SVS IV gene, approximately 3 million plaques of the A Charon 4A library of rat genomic DNA were screened by the Benton-Davis procedure (7), with an amplification step as recommended by S. Woo (8) Electrophoresis of DNA on agarose gels, Southern transfers, and hybridizations were performed as described (10, 11)...

The role of chromatin-dependent regulatory mechanisms in the repression of glucocorticoid-dependent transcription from the murine mammary tumor virus (MMTV) promoter by p65 and E1A was investigated by using chromatin and transiently transfected reporters. The p65 RelA subunit of NF-B represses MMTV expression on either transient or integrated reporters. In contrast, the viral oncoprotein E1A represses a transient but not an integrated MMTV. E1A repression is attenuated by chromatin, suggesting p65 but not E1A manipulates chromatin appropriately to inhibit the GR. Coexpression of p65 and E1A additively represses the transient MMTV but restores the transcriptional activation of the chromatin MMTV in response to glucocorticoids. This indicates that E1A has a dominant chromatindependent activity that attenuates repression by p65. E1A, p65, and GR bind the MMTV promoter, and chromatin remodeling enhances binding on both repressed and activated promoters. In addition, p65 requires Brg for repression of the integrated MMTV. This suggests that neither p65 repression nor E1A attenuation of repression results from an inhibition of remodeling that prevents transcription factor binding. Furthermore, p300/CBP is also required for both repression and attenuation by p65 and E1A. E1A and p65 mutants that do not bind p300/CBP are inactive, indicative of a requirement for p300/CBP-dependent complex formation for both repression and attenuation with chromatin. These data suggest that both the p65-dependent repression and the E1A-mediated attenuation of repression require the Brg1-dependentchromatinremodelingfunctionandp300/CBPdependent complex formation at a promoter assembled within chromatin.

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