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1.
Cloning of DNA complementary to cytochrome P-450 induced by pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and induction response 总被引:10,自引:0,他引:10
The major form of cytochrome P-450 (P-450PCN) was isolated from rats administered pregnenolone-16 alpha-carbonitrile (PCN). Messenger RNA coding for P-450PCN was enriched by polysome immunoadsorption and utilized to construct a library of cDNA clones in pBR322. P-450PCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-450PCN immunoenriched poly(A) RNA. The P-450PCN clone with the largest cDNA insert (pP450PCN-10) was verified to contain sequences complementary to P-450PCN mRNA by hybrid selection-translation. pP450PCN-10 was composed of approximately 1900 base pairs and had a restriction map that overlapped at least 3 other cDNA clones selected by differential colony hybridization. Denaturing-agarose gel electrophoresis and nitrocellulose blot-hybridization using nick-translated 32P-labeled pP450PCN-10 indicated that pP450PCN mRNA is 2500 +/- 150 nucleotides in length; pP450PCN-10, therefore, represents approximately 76% of its corresponding mRNA sequence. Southern blot analysis of rat DNA using pP450PCN revealed that approximately 50 to 60 kilobases of DNA reacted with the PCN probe, suggesting the P-450PCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseudogenes or related P-450 genes that share homology. The mechanism of P-450PCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-450PCN mRNA using pP450PCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-450PCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cytochrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approximately 2-fold) after PCN administration. 相似文献
2.
The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences 总被引:24,自引:0,他引:24
Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site. 相似文献
3.
Complete cDNA and protein sequence of a pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450. A representative of a new gene family 总被引:18,自引:0,他引:18
F J Gonzalez D W Nebert J P Hardwick C B Kasper 《The Journal of biological chemistry》1985,260(12):7435-7441
A full-length cDNA complementary to rat liver mRNA coding for pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 (P-450PCN) was isolated and completely sequenced. P-450PCN mRNA is 2038 nucleotides in length and has a continuous reading frame (82-1596) that encodes a protein of 504 amino acids (Mr = 57,917). The amino-terminal sequence of 18 residues of the purified P-450PCN protein agrees with the open reading frame of the cDNA sequence. The P-450PCN mRNA nucleotide and amino acid sequences clearly establish that this cytochrome is a member of a separate P-450 family different from the phenobarbital-induced (e.g. P-450e) and 3-methyl-cholanthrene-induced (e.g. P-450c) P-450 gene families. P-450PCN shares 38 and 37% nucleotide similarity and 33 and 33% amino acid similarity with P-450e and P-450c, respectively. P-450PCN, P-450e, and P-450c exhibit greater homology in the C-terminal half than in the N-terminal half of the proteins. Included in this region is the cysteinyl fragment (surrounding residue 443 in P-450PCN), which appears to be the most conserved among all fragments of other P-450 proteins. Of interest, the N-terminal region of P-450PCN does not contain the cysteine residue previously thought to contribute the thiolate ligand to the heme iron in P-450 proteins; these data establish more firmly the cysteine residue located in the carboxylterminal region as serving this function. These sequence studies further support the conclusion derived from chromosomal localization studies and Southern blot analyses that P-450PCN represents a member of a distinct third family of P-450 genes, which diverged from a common ancestor more than 200 million years ago. 相似文献
4.
A gene structure of testosterone 6 beta-hydroxylase (P450IIIA) 总被引:3,自引:0,他引:3
M Miyata K Nagata Y Yamazoe R Kato 《Biochemical and biophysical research communications》1991,177(1):68-73
Genomic clones of a rat testosterone 6 beta-hydroxylase have been isolated and characterized as the first gene (P450/6 beta A) among P450IIIA subfamily. This gene spans about 25Kb and consists of 13 exons, which is the largest number of exons among cytochrome P-450 genes reported previously. The nucleotide sequence of the exon region showed high similarity to those of P450PCN2 and P450PCN1 cDNA (Gonzalez, F.J. et al. (1987) Mol. Cell. Biol. 2969-2974), but several replacements and deletions of nucleotide were found between the P450/6 beta A gene and both cDNAs, indicating the existence of multiple P450IIIA genes in rats. 相似文献
5.
Regulation of mouse cytochrome P3-450 by the Ah receptor. Studies with a P3-450 cDNA clone 总被引:4,自引:0,他引:4
The Ah locus in the C57BL/6N mouse regulates at least two cytochrome P-450 gene products, termed in the mouse P1-450 and P3-450; these two enzymes are so named because each is responsible for the highest turnover number for the substrates benzo[a]pyrene and acetanilide, respectively. A cDNA library was prepared in pBR322 from sucrose gradient fractionated total liver poly(A+)-enriched RNA (approximately 20 S) from 2,3,7,8-tetrachlorodibenzo-p-dioxin- (TCDD) treated C57BL/6N (Ahb/Ahb) mice. Differential colony hybridization screening, with [32P]cDNA probes derived from total liver mRNA of both TCDD-treated and control C57BL/6N mice, yielded pP(3)450-21 (1710 base pair) and pP(1)450-57 (1770 base pair) cDNA clones. pP(1)450-57 was found to have 690 base pairs 5'-ward of the original P1-450 cDNA cloned in this laboratory. Restriction maps of pP(3)450-21 and pP(1)450-57 are markedly different and clearly are derived from separate genes. By means of hybridization-translation-arrest experiments, anti-(P3-450) precipitates the translation product (Mr approximately equal to 55000) of mRNA specifically hybridizing to pP(3)450-21. It is also shown that hybridization-translation-arrest experiments using polyclonal antibodies are not specific for proof of a P-450 cDNA clone. pP(3)450-21 was used to probe liver mRNA from Ahb/Ahb, Ahb/Ahd, and Ahd/Ahd mice treated with 3-methylcholanthrene, beta-naphthoflavone, aroclor 1254, isosafrole, low TCDD, or high TCDD. These genetic data rigorously demonstrate control of the P3-450 (20S) mRNA induction process by the Ah receptor. pP(3)450-21 fragments hybridized to TCDD-induced C57BL/6N mRNA and to a portion of the cloned 5' end of the P1-450 gene from a mouse MOPC 41 plasmacytoma library.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
We have examined the tissue-specific expression of three rabbit genes that are closely related members of a subfamily of the phenobarbital-inducible cytochrome P-450 gene family. Analysis of the levels of mRNA in liver revealed that (a) cytochrome P-450PBc1 mRNA was not detectable in livers from control animals but was present in livers from animals treated with phenobarbital, (b) cytochrome P-450PBc2 was present in control tissue and was increased by about 3-fold 24 h after phenobarbital treatment, and (c) the levels of cytochrome P-450PBc3 mRNA was the same in livers from control and treated animals. In the kidney, only P-450PBc2 mRNA was detected at a level 15% of that in the liver, and the levels increased about 3-fold after phenobarbital treatment. None of the mRNAs was detected in lung tissue. Multiple species of RNA were observed that hybridized to probes for cytochrome P-450PBc1 and P-450PBc2 cDNAs by Northern blot analysis ranging in size from 2300 to 4000 nucleotides. Differential sites for polyadenylation probably cause the heterogeneity in size. A single species of RNA of 2200 nucleotides that hybridized to cytochrome P-450PBc3 cDNA probes was observed. These data demonstrate that three closely related cytochrome P-450 genes are differentially responsive to phenobarbital treatment and that they exhibit different tissue-specific patterns of expression. 相似文献
7.
Constitutive testosterone 6 beta-hydroxylase in rat liver 总被引:1,自引:0,他引:1
The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB. 相似文献
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12.
Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes 总被引:2,自引:0,他引:2
H J Burger E G Schuetz J D Schuetz P S Guzelian 《Archives of biochemistry and biophysics》1990,281(2):204-211
We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or "phenobarbital-like" inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis. 相似文献
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Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p 总被引:17,自引:0,他引:17
S A Wrighton P Maurel E G Schuetz P B Watkins B Young P S Guzelian 《Biochemistry》1985,24(9):2171-2178
We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p. 相似文献
15.
cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6 总被引:5,自引:0,他引:5
D Pompon 《European journal of biochemistry》1988,177(2):285-293
A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed. 相似文献
16.
A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment. 相似文献
17.
E G Schuetz S A Wrighton J L Barwick P S Guzelian 《The Journal of biological chemistry》1984,259(3):1999-2006
We administered a series of steroid hormones to primary nonproliferating cultures of adult rat hepatocytes and found that dexamethasone and other glucocorticoids but not sex steroid hormones, mineralocorticoids, or derivatives of pregnenolone other than pregnenolone 16 alpha-carbonitrile (PCN) stimulated de novo synthesis of an immunoreactive protein, indistinguishable from the form of cytochrome P-450 (P450PCN) induced by PCN in rat liver. No difference were discerned among purified liver cytochromes from rats treated with dexamethasone, PCN or dexamethasone plus PCN, among proteolytic digests of these proteins, or among the immunoprecipitated cytochromes prepared from cultured hepatocytes treated with these steroids as judged by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate followed by immunoblot analysis. Of the steroids tested, dexamethasone proved to be the most efficacious inducer increasing the rate of synthesis of P450PCN from 0.05% of total cellular protein synthesis in incubated control cultures (measured as incorporation of [3H]leucine into immunoprecipitable P450PCN) to as much as 9.4% in cultures incubated for 5 days in medium containing dexamethasone (10(-5) M). As with traditional glucocorticoid-responsive liver functions, induction of immunoreactive P450PCN was dependent on the concentration of dexamethasone (10(-8) to 10(-5) M) and was promptly reversed upon withdrawal of the steroid. However, during the 24-h interval between 24 to 48 h of culture age the hepatocytes were refractory to either induction or de-induction of immunoreactive P450PCN even though continuous exposure of the cells to dexamethasone (including this interval) was mandatory for maximal induction of P450PCN at 120 h in culture. Unlike cultured rat hepatocytes, HTC hepatoma cultures failed to exhibit dexamethasone-responsive expression of immunoreactive P450PCN. We conclude that glucocorticoids and PCN constitute a specific "class" of synthetic and endogenous inducers of a single form of cytochrome P-450. 相似文献
18.
A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone. 相似文献
19.
F J Gonzalez S Kimura B J Song J Pastewka H V Gelboin J P Hardwick 《The Journal of biological chemistry》1986,261(23):10667-10672
20.
Recently we reported that a P-450c27/25 cDNA probe hybridizes to two RNA species of about 1.9 and 2.3-2.4 kilobase pairs (kb) in some rat tissues. To understand the molecular relationship between the two mRNAs, we have isolated and characterized a cDNA for the larger, previously uncharacterized 2.3-kb mRNA species. The 2.3-kb cDNA is identical to the previously reported 1.9-kb P-450c27/25 cDNA excepting a 400-nucleotide-long 5' extension. The terminal 291 nucleotides of this extension exhibit 100% complementarity with the 5'-translated region of the mRNA belonging to a family of growth hormone-inducible serine protease inhibitors (SPI). Northern blot analysis, using strand-specific probes, and S1 nuclease protection revealed the presence of the 2.3-kb mRNA exhibiting the sequence characteristics of the larger cDNA. These results were further confirmed by polymerase chain reaction amplification of reverse transcribed RNA. Expression of the 2.3-kb cDNA in COS cells resulted in the correct mitochondrial targeting of a 52-kDa protein exhibiting the properties of P-450c27/25. Furthermore, both the 1.9- and 2.3-kb mRNAs appear to direct the synthesis of a similarly sized 55-kDa precursor protein in a reticulocyte lysate system. Restriction mapping, polymerase chain reaction amplification and partial sequencing of a 25-kb genomic DNA clone suggest the proximal location of the SPI and the P-450c27/25 protein coding regions in the rat genome on either side of a common overlap region. The results also show that the P-450c27/25 mRNAs are regulated by growth hormone in parallel to the SPI mRNAs. These results collectively suggest that a growth hormone-inducible SPI family mRNA and the P-450c27/25 mRNA are encoded by two closely linked, possibly overlapping genes. 相似文献