A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

The upstream open reading frame mediates constitutive effects on translation of cytochrome p-450c27 from the seventh in-frame AUG codon in rat liver

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55-kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression.     

Characterization of a second gene product related to rabbit cytochrome P-450 1

A cDNA, p1-88, was cloned from a library constructed using rabbit liver mRNA. Sequence analysis indicates that p1-88 is highly similar (congruent to 95%) to the cDNA, p1-8, that encodes rabbit liver cytochrome P-450 1 and that had been isolated from the same library. The predicted amino acid sequence of the protein encoded by p1-88, P-450 IIC4, differs at 25 of 487 amino acids from that encoded by p1-8. P-450 IIC4 was synthesized in vitro using rabbit reticulocyte lysate primed with RNA transcribed from the coding sequence of p1-88 using a bacteriophage T7 RNA polymerase/promoter system. P-450 IIC4 reacts with two monoclonal antibodies that recognize P-450 1 and exhibits the same relative electrophoretic mobility as P-450 1. In contrast, the reactivity of a third monoclonal antibody recognizing P-450 1, 1F11, toward P-450 IIC4 synthesized in vitro is greatly diminished. The latter antibody extensively inhibits hepatic progesterone 21-hydroxylase activity and recognizes phenotypic differences among rabbits in the microsomal concentration of P-450 1. This difference in the immunoreactivity of P-450 IIC4 and P-450 1 with the 1F11 antibody suggests that P-450 IIC4 does not contribute significantly to hepatic progesterone 21-hydroxylase activity. S1 nuclease mapping demonstrates that the expression of mRNAs corresponding to p1-88 are expressed to equivalent extents in rabbits exhibiting high and low expression of mRNAs corresponding to p1-8. Thus, P-450 1 differs from the protein encoded by p1-88, in its regulation, immunoreactivity, and by inference its catalytic properties although the amino acid sequences of P-450 1 and P-450 IIC4 are highly similar (congruent to 95%).     

Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia

Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.     

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.     

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.     

Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.     

Titration of mRNAs for cytochrome P-450c and P-450d under drug-inductive conditions in rat livers by their specific probes of cloned DNAs

By sequence analysis of the cloned cDNA or genomic DNA, we have recently deduced the complete primary structures of two forms of 3-methylcholanthrene-inducible cytochromes P-450 (P-450c and P-450d). Comparing these sequences, we identified two highly conserved regions, amino acid numbers from 35 to 200 and from 340 to 470. The nucleotide sequences corresponding to these homologous regions are also well conserved, whereas other regions have undergone considerable sequence divergence. In RNA blot analysis with unfractionated mRNA isolated from 3-methylcholanthrene-treated rat livers, Probe A (specific to P-450c sequence) hybridized with mRNA around 23 S, while Probe B (specific to P-450d sequence) hybridized with mRNA around 18 S. When common sequence between P-450c and P-450d was used as the probes (Probe C or D), two bands were clearly observed around 23 and 18 S mRNAs. With the common DNA sequence between P-450c and P-450d as a probe (93.7% homology), we studied the induction of specific mRNA for P-450c and P-450d by a single dose of several chemical compounds to rats. 3-Methylcholanthrene increased both P-450c and P-450d mRNA levels by 50 and 10 times above the control at 17 h after the administration, respectively. Despite the lower induction rates, the P-450d mRNA level was constantly higher than or at least similar to that of P-450c mRNA. beta-Naphthoflavone and Kaneclor KC 500 showed similar induction ability to 3-methylcholanthrene. On the other hand, isosafrole induced P-450d mRNA to a much greater extent than P-450c mRNA.     

Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G

The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of distinct mRNAs coding for putative isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Three distinct clones encoding full-length 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic-AMP-dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N-terminal portion of 10 amino acids. Poly(A)-rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N-terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK-2/FBPase-2 mRNAs. There were a 6.8-kb mRNA typical of cardiac tissue, a 2.1-kb mRNA typical of liver, corresponding to clone 22c, and a 1.9-kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5'-untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK-2/FBPase-2 isozymes.     

Complete cDNA and protein sequence of a pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450. A representative of a new gene family

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.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment

Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs.