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• W2017242560 abstract "hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7α-hydroxylase (CYP7) and contains a postulated steroidogenic domain present in other steroid-metabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids (neurosteroids) to modulate cognitive function in vivo. hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7α-hydroxylase (CYP7) and contains a postulated steroidogenic domain present in other steroid-metabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids (neurosteroids) to modulate cognitive function in vivo. INTRODUCTIONCytochromes P450, a diverse group of heme-containing monooxygenases (termed CYPs) 1The abbreviations used are: CYPcytochrome P450 (for simplicity no systematic distinction is made between genes encoding mouse (Cyp), rat, and human (CYP) orthologs or their protein products)LTPlong term potentiationNMDAN-methyl-D-aspartatekbkilobase(s)ntnucleotide(s). (for nomenclature see Nelson et al.(1.Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1647) Google Scholar)), catalyze a variety of oxidative conversions, notably of steroids but also of fatty acids and xenobiotics. Though most abundantly expressed in the testis, ovary, placenta, adrenal, and liver, the brain is a further site of CYP expression(2.Walther B. Ghersi-Egea J.F. Minn A. Siest G. Brain Res. 1986; 375: 338-344Crossref PubMed Scopus (99) Google Scholar, 3.Kapitulnik J. Gelboin H.V. Guengerich F.P. Jacobowitz D.M. Neuroscience. 1987; 20: 829-833Crossref PubMed Scopus (81) Google Scholar, 4.Warner M. Strömstedt M. Wyss A. Gustafsson J.A. J. Steroid Biochem. Mol. Biol. 1993; 47: 191-194Crossref PubMed Scopus (34) Google Scholar, 5.Bhamre S. Anandatheerathavarada H.K. Shankar S.K. Boyd M. Ravindranath V. Arch. Biochem. Biophys. 1993; 301: 251-255Crossref PubMed Scopus (36) Google Scholar, 6.Warner M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1019-1023Crossref PubMed Scopus (121) Google Scholar). Several CYP activities or mRNAs have been reported in the nervous system, predominantly of types metabolizing fatty acids and xenobiotics (subclasses CYP2C, −2D, −2E, and −4(6.Warner M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1019-1023Crossref PubMed Scopus (121) Google Scholar, 7.Strömstedt M. Warner M. Gustafsson J.A. J. Neurochem. 1994; 63: 671-676Crossref PubMed Scopus (35) Google Scholar)). However, primary rat brain-derived glial cells can synthesize pregnenolone and progesterone in vitro(8.Jung-Testas I. Hu Z.Y. Baulieu E.E. Robel P. Endocrinology. 1989; 125: 2083-2091Crossref PubMed Scopus (351) Google Scholar). Mellon and Deschepper (9.Mellon S.H. Deschepper C.F. Brain Res. 1993; 629: 283-292Crossref PubMed Scopus (354) Google Scholar) provided molecular evidence for the presence, in brain, of key steroidogenic enzymes CYP11A1 (scc) and CYP11B1 (11β) but failed to detect CYP17 (c17) or CYP11B2 (AS). Though CYP21A1 (c21) activity is reported to be present in brain (10.Casey M.L. MacDonald P.C. Endocr. Rev. 1982; 3: 396-403Crossref PubMed Scopus (81) Google Scholar) authentic CYP21A1 transcripts were not detected(11.Chung B. Matteson K.J. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4243-4247Crossref PubMed Scopus (89) Google Scholar).Interest in brain steroid metabolism has been fueled by the finding that adrenal- and brain-derived steroids (neurosteroids) can modulate cognitive function and synaptic plasticity (reviewed in (12.McEwen B.S. De Kloet E.R. Rostene W. Physiol. Rev. 1986; 66: 1121-1188Crossref PubMed Scopus (1120) Google Scholar, 13.Baulieu E.E. Biol. Cell. 1991; 71: 3-10Crossref PubMed Scopus (282) Google Scholar, 14.Filipini D. Gijsbers K. Birmingham K. Kraulis I. Dubrovsky B. J. Steroid Biochem. Mol. Biol. 1991; 39: 245-252Crossref PubMed Scopus (23) Google Scholar, 15.McEwen B.S. Trends Endocrinol. Metab. 1991; 2: 62-67Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 16.Dubrovsky B. Gijsbers K. Filipini D. Birmingham M.K. Cell. Mol. Neurobiol. 1993; 13: 399-414Crossref PubMed Scopus (37) Google Scholar, 17.Costa E. Auta J. Guidotti A. Korneyev A. Romeo E. J. Steroid Biochem. Mol. Biol. 1994; 49: 385-389Crossref PubMed Scopus (91) Google Scholar)). For instance, pregnenolone and steroids derived from it are reported to have memory-enhancing effects in mice(18.Flood J.F. Smith G.E. Roberts E. Brain Res. 1988; 447: 269-278Crossref PubMed Scopus (212) Google Scholar, 19.Flood J.F. Morley J.E. Roberts E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1567-1571Crossref PubMed Scopus (439) Google Scholar). However, the full spectrum of brain CYPs and the biological roles of their metabolites in vivo have not been established.To investigate such regulation of brain function our studies have focused on the hippocampus, a brain region important in learning and memory. Patients with lesions that include the hippocampus display pronounced deficits in the acquisition of new explicit memories; in rat, neurotoxic lesions to the hippocampus lead to a pronounced inability to learn a spatial navigation task, such as the water maze (20.Morris R.G.M. Garrud P. Rawlins J.N.P. Nature. 1982; 297: 681-683Crossref PubMed Scopus (5026) Google Scholar). Hippocampal synapses, notably those in region CA1, display a particularly robust form of activity-dependent plasticity known as long term potentiation (LTP) (21.Bliss T.V.P. L⊘mo T. J. Physiol. (Lond.). 1973; 232: 331-356Crossref Scopus (4820) Google Scholar) that satisfies some of the requirements for a molecular mechanism underlying memory processes, persistence, synapse specificity, and associativity. LTP is thought to be initiated by calcium influx through the NMDA (N-methyl-D-aspartate) subclass of receptor activated by the excitatory neurotransmitter, L-glutamate (reviewed in (22.Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9458) Google Scholar)); occlusion of NMDA receptors with AP5 both blocks LTP and the acquisition of the spatial navigation task(23.Morris R.G.M. Anderson E. Lynch G. Baudry M. Nature. 1986; 319: 774-776Crossref PubMed Scopus (2755) Google Scholar). In vivo, simultaneous release of γ-aminobutyric acid from inhibitory interneurons inhibits NMDA channel opening and LTP induction(22.Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9458) Google Scholar). It is of note that some naturally occurring steroids, such as pregnenolone sulfate, act as agonists of the γ-aminobutyric acid receptor (e.g. see (24.Harrison N.L. Majewska M.D. Harrington J.W. Barker J.L. J. Pharmacol. Exp. Ther. 1987; 241: 346-353PubMed Google Scholar) and (25.Turner J.P. Simmonds M.A. Br. J. Pharmacol. 1989; 96: 409-417Crossref PubMed Scopus (71) Google Scholar)) and may also directly modulate NMDA currents(26.Wu F.S. Gibbs T.T. Farb D.H. Mol. Pharmacol. 1991; 40: 333-336PubMed Google Scholar, 27.Bowlby M.R. Mol. Pharmacol. 1993; 43: 813-819PubMed Google Scholar). Though brain steroids principally appear to exert their effects via the γ-aminobutyric acid and NMDA receptors, there are indications that neurosteroids may also interact with σand progesterone receptors (28.Monnet F.P. Mahé V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (413) Google Scholar, 29.Rupprecht R. Reul J.M.H.M. Trapp T. van Steensel B. Wetzel C. Damm K. Zieglgänsberger W. Holsboer F. Neuron. 1993; 11: 523-530Abstract Full Text PDF PubMed Scopus (278) Google Scholar). However, the pathways of CYP-mediated steroid metabolism in the central nervous system have not been fully elucidated.In addition, non-steroid CYP metabolites also play important roles in brain; CYP-mediated metabolism of psychoactive agents (30.Cholerton S. Daly A.K. Idle J.R. Trends Pharmacol. 1992; 13: 434-439Abstract Full Text PDF PubMed Scopus (263) Google Scholar) and CYP metabolites of arachidonic acid such as prostanoids and eicosanoids (31.Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-735Crossref PubMed Scopus (275) Google Scholar) clearly contribute to the regulation of brain function.As part of a study into the molecular biology of the hippocampal formation and the mechanisms underlying synaptic plasticity, we have sought molecular clones corresponding to mRNA's expressed selectively in the formation. One such cDNA, hct-1 (for hippocampal transcript), was isolated from a cDNA library prepared from adult rat hippocampus. Sequence analysis revealed that hct-1 is a novel cytochrome P450 most closely related to cholesterol- and steroid-metabolizing CYPs but, unlike other CYPs, is predominantly expressed in brain. We present molecular characterization of hct-1 coding sequences from rat and mouse and their expression patterns and discuss the possible role of hct-1 in the central nervous system.MATERIALS AND METHODSPreparation of cDNA LibrariesFollowing anesthesia (sodium pentobarbital) of adult rats (Lister hooded) the hippocampal formation was dissected, including areas CA1-3 and dentate gyrus, subiculum, alvear, and fimbrial fibers but excluding fornix and afferent structures such as septum and entorhinal cortex. The remainder of the brain was also pooled taking care to exclude hippocampal tissue. Total RNAs were prepared by a standard guanidinium isothiocyanate procedure, centrifugation through a CsCl cushion, and poly(A)+ mRNA selected by affinity chromatography on oligo(dT)-cellulose. First strand cDNA synthesis used a NotI adaptor primer (5′-dCAATTCGCGGCCGC(T)15-3′) and Moloney murine leukemia virus reverse transcriptase; second strand synthesis was performed by RNase H treatment, DNA polymerase I fill-in, and ligase treatment. Following the addition of hemiphosphorylated EcoRI adaptors (5′-dCGACAGCAACGG-3′ and 5′-dAATTCCGTTGCTGTCG-3′) and cleavage with NotI the cDNA was inserted between the NotI and EcoRI sites of bacteriophage λ vector λ-ZAPII (Stratagene). The mouse liver cDNA library, also established as NotI-EcoRI fragments in a λ-gt10 vector, was a kind gift of B. Luckow and K. Kästner, Heidelberg.Differential Hybridization ScreeningRecombinant bacteriophage plaques were transferred in duplicate to Hybond-N membranes (Amersham Corp.), denatured (0.5 M NaOH, 1.5 M NaCl, 4 min), renatured (1 M Tris-HCl pH 7.4, 1.5 M NaCl), rinsed, dried, and baked (2 h, 80°C). Hybridization as described (32.Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7258) Google Scholar) used a radiolabeled probe prepared by Moloney murine leukemia virus reverse transcriptase copying of poly(A)+ RNA (from either hippocampus or the remainder of brain) into cDNA in the presence of [α-32P]dCTP and unlabeled dGTP, dATP, and dTTP according to standard procedures. Following washing and exposure for autoradiography, differentially hybridizing plaques were repurified. Inserts were transferred to a pBluescript vector either by cleavage and ligation or by using in vivo excision using the ExAssist/SOLR system (Stratagene).Northern AnalysisTotal RNA was extracted by tissue homogenization in guanidinium thiocyanate according to a standard procedure and further purified by centrifugation through a CsCl cushion. Where appropriate, poly(A)+ RNA was selected on oligo(dT)-cellulose. Electrophoresis of RNA (10 μg) on 1% agarose in the presence of 7% formaldehyde was followed by capillary transfer to nylon membranes, baking (2 h, 80°C), and rinsing in hybridization buffer (0.25 M sodium phosphate, pH 7.2; 1 mM EDTA, 7% SDS, 1% bovine serum albumin) as described(32.Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7258) Google Scholar). Probes were prepared by random priming of DNA polymerase copying of denatured double-stranded DNA. Hybridization (16 h, 68°C) was followed by washing (3 times, 20 mM sodium phosphate, pH 7.2, 1 mM EDTA, 1% SDS, 20 min, 68°C), and membranes were exposed for autoradiography. The loading control probe was a 0.5-kb cDNA encoding the ubiquitously expressed rat ribosomal protein S26(33.Vincent S. Marty L. Fort P. Nucleic Acids Res. 1993; 21: 1498Crossref PubMed Scopus (200) Google Scholar). 2M. Richardson, unpublished data. In Situ HybridizationSynthetic hct-1 oligonucleotide probes 5′-dGACAGGTTTTGTGACCCAAAACAAACTGGATGGATCGCAATC-3′(rat, 55% G + C) and 5′-ATCACGGAGCTCAGCACATGCAGCCTTACTCTGCAAAGCTTC-3′ (mouse, 48% G + C) were labeled using terminal transferase (Boehringer Mannheim) and α-35S-dATP (Amersham) according to the manufacturer's instructions. The control probe, 5′-dAGCCTTCTGGGTCGTAGCTGACTCCTGCTGCTGAGCTGCAACAGCTTT-3′ (56% G + C) was based on human opsin cDNA(34.Nathans J. Thomas D. Hogness D.S. Science. 1986; 232: 193-202Crossref PubMed Scopus (1168) Google Scholar). Frozen coronal 10-μm sections of brain were fixed (4% paraformaldehyde, 10 min), rinsed, treated with proteinase K (20 μg/ml in 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 min), rinsed, and refixed with paraformaldehyde as before. Following acetylation (0.25% acetic anhydride, 10 min) and rinsing, sections were dehydrated by passing though increasing ethanol concentrations (30, 50, 70, 85, 95, 100, and 100%, each for 1 min except the 70% step (5 min)). Following CHCl3 treatment (5 min) and rinsing in ethanol, sections were dried before hybridization. Hybridization in buffer (4 × standard saline citrate (1 × SSC = 0.15 M NaCl, 0.015 M Na3citrate), 50% v/v formamide, 10% w/v dextran sulfate, 1 × Denhardt's solution, 0.1% SDS, 500 μg/ml denatured salmon sperm DNA, 250 μg/ml yeast tRNA) was for 16 h at 37°C. Slides were washed (4 × 15 min, 1 × SSC, 60°C; 2 × 30 min, 1 × SSC, 20°C), dipped into photographic liquid emulsion (LM-1, Amersham Corp.), exposed, and developed according to the manufacturer's specifications. Slides were counterstained with 1% methyl green.DNA Sequence CharacterizationDideoxy sequencing of cDNAs in pBluescript II KS and SK vectors was performed using the Sequenase 2.0 kit (Amersham Corp.) according to the manufacturer's instructions. To sequence larger cDNAs sequential exonuclease III deletions were produced (Erase-a-base, Promega Biotech). Sequence data, obtained for both strands of the larger hct-1 cDNAs, were analyzed using the University of Wisconsin Genetics Computer Group package (UWGCG Version 7, 1991).Southern HybridizationGenomic DNA prepared from mouse or rat liver or from human lymphocytes was digested with the appropriate restriction endonuclease, resolved by agarose gel electrophoresis (0.7%), and transferred to Hybond-N membranes. Following baking (2 h, 80°C), hybridization conditions were as described for Northern analysis.RESULTSDifferential Screening of a Rat Hippocampus cDNA LibraryTo identify genes whose expression is enriched in the hippocampal formation we performed a differential hybridization screen of a hippocampal cDNA library. Adult rat hippocampal RNA was converted to double-stranded cDNA and inserted as EcoRI-NotI fragments into a bacteriophage λvector. Duplicate lifts from 500,000 plaques were screened with radiolabeled cDNA probes prepared from either hippocampus or “rest of brain.” Some 360 clones gave a substantially stronger hybridization signal with the hippocampus probe than with the rest of brain probe; 49 were analyzed in more depth. In vivo excision was used to transfer the inserts to a plasmid vector for partial DNA sequence studies. Of these, 21 were novel (not presented here); others were known genes whose expression is enriched in hippocampus but not specific to the formation (e.g. the rat amyloidogenic protein(35.Shiver B.D. Hilbich C. Multhaur G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (387) Google Scholar)). Northern analysis was first performed using radiolabeled probes corresponding to the 21 novel sequences. While three (12.10a, 14.5a, and 15.13a) identified transcripts specific to the hippocampus, 12.10a and 15.13a both hybridized to additional transcripts whose expression was not restricted to the formation (not presented). Clone 14.5a appeared to identify transcripts enriched in hippocampus (see below) and was dubbed hct-1.Rat hct-1 Encodes a Cytochrome P450The insert of clone 14.5a (300 nt) was used to rescreen the hippocampal cDNA library. Four positives were identified (clones 14.5a-5, −7, −12, and −13), and the region adjacent to the poly(A) tail was analyzed by DNA sequencing. While clones 5 (0.7 kb) and 12 (1.4 kb) had the same 3′ end as the parental clone, clone 7 (0.9 kb) had a different 3′ end consistent with utilization of an alternative polyadenylation site (see below). Clone 13 (2.5 kb), dubbed hct-2, appeared unrelated to hct-1 (not presented).Clones 12 and 7 were then fully sequenced and compared with the data base. Homology was detected between clone 12 and the human (36.Noshiro M. Okuda J.Y. FEBS Lett. 1990; 268: 137-140Crossref PubMed Scopus (93) Google Scholar) and rat (37.Noshiro M. Nishimoto M. Morohashi K. Okuda K. FEBS Lett. 1989; 257: 97-100Crossref PubMed Scopus (73) Google Scholar, 38.Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar) cDNA's encoding cholesterol 7α-hydroxylase (CYP7), though the sequences are clearly distinct. At the nucleic acid level, the 1428-nt cDNA clone for rat hct-1 shared 55% identity over an 1100-nt overlap with human CYP7 and 54% identity over a 1117-nt overlap with rat CYP7 (not presented). Fig. 1 gives the partial cDNA sequences of rat hct-1 and the encoded polypeptide.hct-1 mRNA Expression in RatRat hct-1 clone 14.5a/12 (1.4 kb) was used to investigate the expression of hct-1 mRNA in rat brain and other organs. While preliminary in situ hybridization experiments did not permit unambiguous localization of hct-1 transcripts, we confirmed expression in the hippocampus, predominantly in the cell layers of the dentate gyrus, with weaker expression in other hippocampal and brain regions (not presented). Upon Northern analysis (Fig. 2A) the hct-1 probe identified three transcripts in hippocampus of 5.0, 2.1, and 1.8 kb, with the two smaller transcripts being particularly enriched in hippocampus. The larger transcript was only detectable in brain, while the two smaller transcripts were also present in liver (and, at much lower levels, in kidney) but were not detected in other organs tested including adrenal (not shown), testis, and ovary. In brain, expression was also detected in olfactory bulb and cortex while very low levels were present in cerebellum (Fig. 2A). Whereas the 1.8- and 2.1-kb transcripts are thought to derive from alternative utilization of polyadenylation sites within hct-1 transcripts, we were unable to confirm that the 5.0-kb transcript is encoded by the same gene because we failed to isolate correspondingly large cDNAs from the rat brain library and an equivalent 5-kb transcript was not detected in mouse (see below).Figure 2:Northern analysis of hct-1 expression in adult rat and mouse brain. A, expression in rat brain and other tissues; B, sexually dimorphic expression in rat liver; C, expression in mouse tissues. Poly(A)+ (A) or total (B and C) RNA from organs of adult animals was resolved by gel electrophoresis; the hybridization probe was rat hct-1 cDNA clone 12 (1.4 kb); the probe for the loading control (below) corresponds to ribosomal protein S26. Tissues analyzed were: Hi, hippocampus; RB, remainder of brain lacking hippocampus; Cx, cortex; Cb, cerebellum; Ob; olfactory bulb; Li, liver; He, heart; Th, thymus; Ki, kidney; Ov, ovary; Te, testis; Lu, lung.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sexual Dimorphism of hct-1 Expression in Liver but Not in BrainThe expression of several CYPs is known to be sexually dimorphic in liver (reviewed by Morgan and Gustafsson(39.Morgan E.T. Gustafsson J.A. Steroids. 1987; 49: 213-245Crossref Scopus (10) Google Scholar)). We therefore inspected liver and brain of male and female rats for the presence of hct-1 transcripts. In Fig. 2B the hct-1 probe revealed the 1.8-, 2.1-, and 5.0-kb transcripts in both male and female brain, with the 2.1-kb hct-1 transcript predominating. While levels of hct-1 mRNAs in liver were reduced greater than 20-fold over those detected in brain, hct-1 transcripts were only significant in liver from male animals; expression in females was barely detectable, demonstrating that hepatic expression of hct-1 is sexually dimorphic. The 5.0-kb transcript was not detected in liver.Isolation of Mouse hct-1 cDNA ClonesBecause the hct-1 transcripts identified (predominantly 1.8 and 2.1 kb) are longer than the largest cDNA clone (1.4 kb) obtained from our rat hippocampus library we pursued studies with the mouse hct-1 ortholog. A mouse liver cDNA library was screened using a rat hct-1 probe, and four clones were selected, none containing a poly(A) tail. Two (clones 33 and 35, both 1.8 kb) gave identical DNA sequences at both their 5′ and 3′ ends, and this sequence was approximately 91% similar to rat hct-1 (not shown). The remaining two clones, 23 and 40, were also identical to each other and were related to the other clones except for a 5′ extension (59 nt) and a 3′ deletion (99 nt). The complete DNA sequences of clones 35 and 40 were therefore determined.The sequences obtained were identical throughout the region of overlap. The mouse hct-1 open reading frame commences with a methionine at nucleotide 81 (numbering from clone 40) and terminates with a TGA codon at nucleotide 1600, encoding a protein of 507 amino acids (Fig. 3). At the 5′ end the ATG initiation codon leading the open reading frame does not correspond to the translation initiation consensus sequence (YYAYYATGR(40.Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4151) Google Scholar)). However, the 5′-untranslated region cloned is devoid of other possible initiation codons, and an in-frame termination triplet (TAA) lies 20 codons upstream of the ATG. The encoded polypeptide sequence aligns well with other cytochrome P450 sequences (see below); we surmise that the ATG at position 81 represents the correct start site for translation. At the 3′ end the truncation of clone 40 lies entirely in the non-coding region downstream of the stop codon. Neither clone contained a poly(A) tail, but both contained a potential polyadenylation sequence (AATAAA) at a position corresponding precisely to that seen in the rat cDNA.Figure 3:Mouse hct-1 cDNA and the sequence of the encoded polypeptide. The restriction map of the cDNA (top) corresponds to the compilation of two independent clones sequenced; the cross-hatched box indicates the coding region. The nucleotide sequence and translation product (bottom) are derived from this compilation. Lower case sequences indicate the 59 additional 5′ nucleotides in clone 40 and the 99 additional 3′ nucleotides in clone 35. The putative polyadenylation site is underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structure of Mouse hct-1 PolypeptideAs anticipated, nucleotide sequence homology of mouse hct-1 was highest with human CYP7 (~56% identity over the coding region). At the polypeptide level the mouse open reading frame shows 81% identity to the rat hct-1 polypeptide over 414 amino acids; the overall degree of similarity may be different as the full protein sequence of rat hct-1 is not known. Both the human (CYP7(36.Noshiro M. Okuda J.Y. FEBS Lett. 1990; 268: 137-140Crossref PubMed Scopus (93) Google Scholar)) and rat (37.Noshiro M. Nishimoto M. Morohashi K. Okuda K. FEBS Lett. 1989; 257: 97-100Crossref PubMed Scopus (73) Google Scholar, 38.Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. J. Biol. Chem. 1990; 265: 8190-8197Abstract Full Text PDF PubMed Google Scholar) CYP7 polypeptides share 39% amino acid sequence identity to mouse hct-1. Fig. 4A presents the alignment of mouse hct-1 polypeptide with human CYP7.Figure 4:Alignment of mouse hct-1 with human CYP7 (cholesterol 7α-hydroxylase) and steroidogenic P450 s. A, identical amino acids are indicated by a bar; hyphens in the amino acid sequences indicate gaps introduced during alignment. The N-terminal hydrophobic leader sequences are underlined. The position of the conserved Thr residue within the O2-binding pocket of other CYPs(43.Mornet E. Dupont J. Vitek A. White P.C. J. Biol. Chem. 1989; 264: 20961-20967Abstract Full Text PDF PubMed Google Scholar), but replaced by Asn in hct-1 (position 294) and CYP7, is indicated by an asterisk. B and C, conserved residues in the heme-binding (residues 440-453, B) and postulated steroidogenic (residues 348-362, C) domains conserved between hct-1 and other similar CYPs (overlined in A). Sequences are human CYP7 (7α-hydroxylase(36.Noshiro M. Okuda J.Y. FEBS Lett. 1990; 268: 137-140Crossref PubMed Scopus (93) Google Scholar)), bovine CYP17 (17α-hydroxylase (42.Zuber M.X. John M.E. Okamura T. Simpson E.R. Waterman M.R. J. Biol. Chem. 1986; 261: 2475-2482Abstract Full Text PDF PubMed Google Scholar)), human CYP11B1 (steroid β-hydroxylase(43.Mornet E. Dupont J. Vitek A. White P.C. J. Biol. Chem. 1989; 264: 20961-20967Abstract Full Text PDF PubMed Google Scholar)), bovine CYP21B (21-hydroxylase(11.Chung B. Matteson K.J. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4243-4247Crossref PubMed Scopus (89) Google Scholar)), human CYP11A1 (P450scc; cholesterol side chain cleavage(44.Morohashi K. Sogawa K. Omura T. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1987; 101: 879-887Crossref PubMed Scopus (158) Google Scholar)), and rabbit CYP27 (27-hydroxylase(45.Andersson S. Davis D.L. Dahlbäck H. Jörnvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar)).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The N terminus of the hct-1 polypeptide is hydrophobic, a feature shared by microsomal CYPs. This portion of the polypeptide is thought to insert into the membrane of the endoplasmic reticulum. Consistent with microsomal CYPs, the N terminus lacks basic amino acids prior to the hydrophobic core (amino acids 9-34). Previous alignment studies have highlighted conserved regions within CYP polypeptides (e.g.(46.Nelson D.R. Strobel H.W. J. Biol. Chem. 1988; 263: 6038-6050Abstract Full Text PDF PubMed Google Scholar)). CYPs contain a highly conserved motif, FXXGXXXCXG(XXXA), present in 202 of the 205 compiled sequences(1.Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1647) Google Scholar), thought to represent the heme binding site with the arrangement of amino acids around the cysteine residue postulated to preserve the three-dimensional structure of thi" @default.
• W2017242560 created "2016-06-24" @default.
• W2017242560 creator A5002375093 @default.
• W2017242560 creator A5002665222 @default.
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• W2017242560 date "1995-12-01" @default.
• W2017242560 modified "2023-10-15" @default.
• W2017242560 title "A Novel Cytochrome P450 Expressed Primarily in Brain" @default.
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