Biomedical subjects

K Sogawa

Publications and source records attributed to K Sogawa.

At least 109 records · Page 6Linked to original sources

PubMed1988

Hypomethylation and expression of pepsinogen A genes in the fundic mucosa of human stomach.

We have examined the correlation between the extents of methylation and expression of pepsinogen A genes in normal human tissues. Expression of pepsinogen A mRNA was detected only in the fundic mucosa of the stomach and both CCGG and GCGC sites in the genes region were less methylated in the fundic mucosa than in other non-expressing tissues. Thus, there was an inverse correlation between the extents of methylation and expression of pepsinogen A genes and the role of DNA methylation in the regulation of pepsinogen A genes expression during normal differentiation was suggested.

DNA↗

PubMed1988

Primary structure of human pepsinogen C gene.

The entire human pepsinogen C gene has been isolated from a cosmid genomic library. The nucleotide sequences of all the exons and the 5'- and 3'-flanking regions of the gene are presented. The organization of the gene is fundamentally compatible with those of other aspartic proteinases, allowing us to conclude that the genes of these aspartic proteinases including pepsinogen C are derived from a common ancestral gene. The predicted 388-residue amino acid sequence of human pepsinogen C consists of a signal sequence of 16-amino acid residues, an activation peptide of 43 residues, and the mature pepsin of 329 residues containing the two active-site aspartic acids. In the light of present notions about eukaryotic gene expression, possible regulatory roles of the oligonucleotide DNA sequences in the promoter region of the gene are discussed.

Amino Acid Sequence↗

PubMed1988

Complementary DNA cloning of cytochrome P-450s related to P-450(M-1) from the complementary DNA library of female rat livers. Predicted primary structures for P-450f, PB-1, and PB-1-related protein with a bizarre replacement block and their mode of transcriptional expression.

Three kinds of cDNA clones were isolated from a cDNA library synthesized from female rat liver mRNA by cross-hybridization with the P-450(M-1) cDNA as a probe and sequenced. One clone appears to be the previously isolated P-450f cDNA clone with an additional 5'-untranslated and coding sequence which are lacking in the previously reported clone (Gonzalez, F. J., Kimura, S., Song, B.-J., Pastewka, J., Gelboin, H. V., and Hardwick, J. P. (1986) J. Biol. Chem. 261, 10667-10672), though several nucleotide differences were seen. Another one is for P-450PB-1 mRNA previously isolated, and the last has an almost identical nucleotide sequence to P-450PB-1 (the same report cited above) except for one region of 159 base pairs where the sequence homology between the two is abruptly broken down. This nonhomologous region appears to correspond exactly to the entire eighth exon, estimated by comparison with the gene structure of the related P-450 (P-450(M-1)). This replacement in P-450PB-1 (ps) causes a frameshift in the open reading frame, resulting in the generation of a truncated form of P-450 with a strange replacement block and lacking the heme-binding region. This observation suggests that the mRNA whose cDNA was cloned here was produced from a recombinant gene generated by gene conversion or from alternative splicing of a cryptic exon. Sex- and age-dependent expression of the mRNAs investigated by dot blot analysis revealed that normal- and pseudo-type PB-1 mRNA were expressed in both male and female rat livers, though their age-dependent expression was different in male and female animals. In addition, both the mRNAs were specifically expressed in the female brain of 8 weeks, whereas practically no expression was observed in kidney and lung of both sexes.

Amino Acid Sequence↗

PubMed1987

Gene structure of cytochrome P-450(M-1) specifically expressed in male rat liver.

Cytochrome P-450(M-1) [P-450(M-1)] is specifically expressed in adult male rat liver [Yoshioka, H., Morohashi, K., Sogawa, K., Miyata, T., Kawajiri, K., Hirose, T., Inayama, S., Fujii-Kuriyama, Y., & Omura, T. (1987) J. Biol. Chem. 262, 1706-1711]. Isolation and analysis of the gene for P-450(M-1) revealed that the coding region of the gene is interrupted by eight introns and is dispersed over a 35-kilobase pair region of chromosomal DNA. Intron insertion sites of the P-450(M-1) gene are located at equivalent positions to those of cytochrome P-450b and P-450e, which are phenobarbital-inducible. Sequence analysis of the 5'-upstream region of the P-450(M-1) gene shows that there is a homologous sequence to glucocorticoid regulatory elements (GRE) identified in glucocorticoid-responsive genes.

Amino Acid Sequence↗

PubMed1987

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2).

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.

Amino Acid Sequence↗

PubMed1987

Characterization of xenobiotic responsive elements upstream from the drug-metabolizing cytochrome P-450c gene: a similarity to glucocorticoid regulatory elements.

The DNA element governing the inducible expression of drug-metabolizing P-450c gene by xenobiotic treatments was investigated by gene transfer methods. A variety of dissected fragments from -844 to -1140bp region which was essential for the inducibility of P-450c gene were placed on the heterologous SV40 promoter for testing the inducibility. Mapping studies in combination with gel retardation assay defined the presence of the two xenobiotic responsive elements (XRE, XRE1, -1007 - -1021bp; XRE2, -1088 - -1092bp) composed of about 15 nucleotides which expressed the enhancer activity in response to xenobiotic inducers. The two XREs share 10 nucleotides in common out of 15 as expressed in the sequence CG/CTG/CC/TTG/CTCACGCT/AA and are arranged in the inverse orientation. They are different from DREs (drug responsive element) proposed previously (Sogawa, K. et al. Proc. Natl. Acad. Sci. 83, 8044-8048 (1986] and expressed a strong enhancer activity in response to 3-methylcholanthrene. The XRE shows a significant homology with glucocorticoid regulatory elements and apparently needs normal functions of a putative xenobiotic receptor for the inducible enhancer activity.

Animals↗

PubMed1987

Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers.

cDNA clones for the P-450(M-1) mRNA, which exhibits a male-specific expression in rat livers, were isolated by using synthetic oligonucleotides as the probes. Sequence analysis of the cDNAs showed that P-450(M-1) mRNA contains 1,853 nucleotides in addition to a poly(A) chain, and a single open reading frame of 1,500 nucleotides encodes a polypeptide of 500 amino acids with a Mr = 57,187. The predicted NH2-terminal sequence of 30 amino acids agrees well with that of the purified protein determined by Edman degradation, and the predicted primary structure included all the partial sequences of six internal peptides of P-450(M-1) obtained by the proteolytic digestion and a conserved amino acid sequence containing a putative heme-binding cysteine, proximate to the COOH terminus of the molecules. P-450(M-1) showed relatively high sequence similarity with P-450b (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797) (52% similarity), P-450-3b (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1985) J. Biol. Chem. 260, 5427-5434) (64%), P-450-1 (Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., and Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354) (74%), P-450PBc1 (Leighton, J. K., DeBrunner-Vossbrinck, B. A., and Kemper, B. (1984) Biochemistry 23, 4598-4603) (71%), while its sequence similarity with 3-methylcholanthrene-inducible P-450c and P-450d is rather low. Consequently, P-450(M-1) could be structurally classified into the phenobarbital-inducible type of P-450 gene family. RNA blot analysis using a synthetic oligonucleotide specific for P-450(M-1) revealed that P-450(M-1) mRNA was expressed exclusively in the livers of mature male rats in a sex-specific manner, but not in other tissues so far examined.

Amino Acid Sequence↗

PubMed1987

Low protein--high energy diet induces repressed transcription of albumin mRNA in rat liver.

The effects of diet at the molecular level were investigated by feeding rats control or protein-deficient diets. Each of the control and the protein-deficient groups was further divided into three subgroups according to the level of energy intake. Liver DNA, RNA and total cellular protein concentrations and serum albumin and albumin mRNA concentrations were determined. Protein deficiency caused a marked inhibition of liver growth but the size of most cells remained normal. The low protein diet caused concurrent decreases in the levels of serum albumin and serum mRNA. However, this effect was observed only with the combination of a low protein diet and normal energy intake. The low protein-low energy diet failed to induce the low serum albumin level. Our findings suggest that the altered balance between at least two factors of protein and energy intake serves as a trigger for several metabolic changes which ultimately regulate specific genes.

Animals↗

PubMed1987

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase.

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.

Base Sequence↗

PubMed1987

Molecular cloning and nucleotide sequence of DNA of mitochondrial cytochrome P-450(11 beta) of bovine adrenal cortex.

cDNA clones of the mRNA for bovine adrenal cytochrome P-450(11 beta) were isolated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11 beta), which consisted of 503 amino acids (Mr: 57,924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11 beta). molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11 beta) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11 beta) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11 beta) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11 beta) and P-450(SCC) was 36%, which is higher than the values between P-450(11 beta) and various microsomal P-450s. An alignment of P-450(11 beta) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hydropathy profiles of P-450(11 beta) and P-450(SCC) were very similar. A definite difference was noticed at the NH2-terminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydrophobic sequence consisting of about 20 amino acids, whereas mitochondrial type had an extension peptide containing many positively changed amino acids.

Adrenal Cortex↗

PubMed1986

Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase.

We have isolated cDNA clones of the mRNA for rat UDP-glucuronosyltransferase that catalyzes the glucuronidation of 4-nitrophenol, by using synthetic oligonucleotides as hybridization probes. The complete nucleotide sequence of the 1,927-base pairs cDNA insert has been determined. With untranslated sequences of 124 and 216 base pairs in the 5'- and 3'-terminal regions, respectively, the cDNA insert contained 1,587 base pairs that encode a complete primary structure of a putative precursor form of 4-nitrophenol UDP-glucuronosyltransferase with a calculated molecular weight of 60,114. The cDNA sequence also indicates the presence of 25 amino acids preceding the sequence determined by microsequence of the isolated protein. This extrapeptide, for the most part, consists of hydrophobic amino acids which are characteristic of the signal peptides as found for secretory proteins and most transmembrane proteins. Furthermore, the deduced amino acid sequence contains a putative halt transfer signal of a hydrophobic segment (residues 487-510), which is flanked on both sides by the peptide segments of highly charged amino acid residues (residues 463-486 and 511-529). These features are consistent with the properties of transmembrane proteins. Specific cDNA probes were used to analyze the induction of the enzyme in rat tissues by treatment with 3-methylcholanthrene. RNA blot analysis showed that 3-methylcholanthrene increased 10- to 15-fold the amount of hybridizable mRNA in liver. The livers and kidneys from 3-methylcholanthrene-treated rats were found to contain almost the same amount of hybridizable mRNA, although the basal level in the kidney was much higher than that of the liver, and the amounts in the lung were much lower than that of the liver and kidney.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence of a nearly full-length cDNA coding for pepsinogen of rat gastric mucosa.

A nearly full-length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5'-non-coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3'-non-coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16-residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C-type pepsinogens and is much more homologous in amino acid sequence with C-type pepsinogens than A-type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.

Amino Acid Sequence↗

PubMed1986

Expression of cytochrome P-450d by Saccharomyces cerevisiae.

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.

Animals↗

PubMed1986

Structure and drug inducibility of the human cytochrome P-450c gene.

A human genomic clone (lambda hP-450mc-1), highly homologous to the rat cytochrome P-450c gene, was isolated and analyzed for the complete nucleotide sequence. The gene structure coincides with that of a recently reported human gene isolated from genomic DNA of a human breast carcinoma cell line, MCF-7 [Jaiswal, A.K., Gonzalez, F.J. & Nebert, D.W. (1985) Nucleic Acids Res. 13, 4503-4520] with notable exceptions in the first intron: a 320-base-pair fragment is inserted and a 650-base-pair fragment is deleted in the gene examined in the present study. The 320-base-pair insert appears to contain a moderately repetitive sequence (approx. 140 copies) in the human genome. The 650-base-pair fragment, present in intron 1 of the reported sequence, is dislocated in the lambda hP-450mc-1 to about 10(4) base pairs upstream from the putative transcription initiation site. The results of Southern blot analysis using human total DNA were compatible with the gene structure of lambda hP-450mc-1. A fusion gene, which was constructed by ligating the 5' flanking region of the gene to the structural gene for prokaryotic chloramphenicol acetyltransferase (CAT), inducibly expressed the CAT activity in mouse Hepa-1 cells in response to administered methylcholanthrene, indicating that the isolated human gene is indeed of methylcholanthrene inducibility.

Acetyltransferases↗

PubMed1986

Close linkage of human chromosomal pepsinogen A genes.

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.

Cloning, Molecular↗

PubMed1986

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex.

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).

Adrenal Cortex↗

PubMed1986

Regulatory DNA elements localized remotely upstream from the drug-metabolizing cytochrome P-450c gene.

We have investigated regulatory DNA elements in the expression of the drug metabolizing P-450c gene of rats. After combining the 5' flanking and upstream untranslated regions of the isolated P-450c gene with structural gene for chloramphenicol acetyltransferase (CAT), the fusion genes were transfected into cultured cells (Hepa-1 and L cells) for the assay of transient expression of CAT activity. CAT activity was expressed inducibly in response to 3-methylcholanthrene only in Hepa-1 cells. At least three regions containing regulatory DNA elements were indentified; one, which is present in the sequence from -44b to -0.2kb immediately upstream of the TATA box, functions in the basal level of transcription, and the other two which were located in the sequence from -0.8kb to -1.0kb and from -1.0kb to -6.3kb, enhance in combination, transcription in response to inducers in a manner independent of their orientation.

Acetyltransferases↗

PubMed1986

Location of regulatory elements responsible for drug induction in the rat cytochrome P-450c gene.

The synthesis of cytochrome P-450c is induced remarkably in cultured cells as well as animal tissues in response to added chemicals such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin. To study this mechanism, we joined the sequence of 5'-flanking and upstream regions of the P-450c gene to the structural gene for chloramphenicol acetyltransferase. The fusion gene was introduced into Hepa-1 cells for the assay of the expressed acetyltransferase activity. At least three cis-acting regulatory regions that are responsible for the inductive expression were determined in the sequences from nucleotide -3674 to -3067, from -1682 to -1429, and from -1139 to -1029, relative to the transcription start site, by external deletion analysis. Further detailed analysis of the region (nucleotides -1139 to -1029) most influential on the inducibility revealed that a regulatory element consisting of 10 base pairs termed a drug regulatory element (DRE) and its homologues were tandemly arranged in this region. The consensus sequence deduced from DREs is 5'-GCNTGAGGCTGGG-3'. The regulatory sequence from nucleotide -1140 to -844 is capable of conferring inducibility on a heterologous promoter in a manner independent of its orientation and distance from the subordinate promoter.

Acetyltransferases↗