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.     

Nucleotide sequence of the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking regions.

A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted Alu dimer repeat was identified further downstream.     

Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.     

Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.     

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.     

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 mono-oxygenases, was purified from a cell suspension culture of the higher plant Catheranthus roseus . Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans -cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identity of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animal species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.     

Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.     

Structural details of the human cytochrome P-450c gene

Aryl hydrocarbon hydroxylase activity is most closely associated with cytochrome P-450c in the rat and cytochrome P1-450 in the mouse. The sequence for the orthologous human gene coding for this enzymatic activity has been determined from several sources: cytochrome P-450c isolated from human embryonic DNA [K. Kawajiri, J. Watanabe, O. Gotoh, Y. Tagashiri, K. Sogawa, and Y. Fujii-Kuriyama (1986) Eur. J. Biochem. 139, 219-225], human lymphocytes in our own laboratory, and cytochrome P1-450 isolated from the established human breast carcinoma cell line, MCF-7 [A.K. Jaiswal, F. J. Gonzalez, and D. W. Nebert (1985) Nucleic Acids Res. 13, 4503-4520]. The data from our laboratory agree well with the sequence derived from human embryonic DNA, but differs significantly from that reported for the gene isolated from MCF-7 cells. Among these differences are a 320-bp insert and a 650-bp deletion in intron 1 relative to the sequence derived from the established cell line. We observe two mRNA species that hybridize to cytochrome P-450c probes, one expected at 2.7 kb and an additional 2.0-kb species. Finally, we note additional hybridization bands in 11% of the population examined by Southern blot analysis, representing either a second rare allele or, more likely, a duplication of at least a portion of the cytochrome P-450c gene.     

The human serum amyloid A locus SAA4 is a pseudogene.

We have isolated the human genomic DNA clone GSAA4 from a size-selected Bgl II library by hybridization to a probe derived from the human serum amyloid A gene GSAA1. Sequencing the 5' end of this clone revealed a region similar to the first exon of gene GSAA1 but with significant nucleotide differences and mutation of the 3' splice site. The restriction map of the GSAA4 clone corresponds to that for the locus "SAA4" recently reported by others. Sequence and hybridization details indicate that the locus in clone GSAA4 is a member of the human serum amyloid A gene family and contains a pseudogene. Isolating GSAA4 completes the collection of clones needed to account for all bands found in blot hybridizations of human DNA using serum amyloid A gene probes.     

人细胞色素c基因在大肠杆菌中的克隆和表达及活性测定

通过PCR方法 ,从人胎儿心肌基因组DNA中得到precytc基因 (有 1个内含子 ) ,剔除内含子后 ,得到细胞色素c基因的编码序列 .测序结果表明 ,该基因与GenBank中报道的人细胞色素c基因核苷酸顺序完全一致 .将其插入原核表达载体pET3a的NdeⅠ和HindⅢ位点之间构建pET3a cytc重组质粒 ,并成功转化入E .coliBL2 1(DE3)中 .经IPTG诱导表达后 ,15 %SDS PAGE分析 ,可观察到1条与细胞色素c蛋白分子量相符的电泳条带 .Western印迹结果显示 ,该条带与小鼠抗人cytc单克隆抗体IgG2b发生特异反应 ,证实为人细胞色素c的前体蛋白 .体外使血红素与该前体蛋白结合生成完整的人细胞色素c蛋白 ,其耗氧量在不同浓度具有与反应时间的线性关系 ,为研究人细胞色素c结构和功能关系奠定了基础     

Human homeo box-containing genes located at chromosome regions 2q31----2q37 and 12q12----12q13

Four human homeo box-containing cDNAs isolated from mRNA of an SV40-transformed human fibroblast cell line have been regionally localized on the human gene map. One cDNA clone, c10, was found to be nearly identical to the previously mapped Hox-2.1 gene at 17q21. A second cDNA clone, c1, which is 87% homologous to Hox-2.2 at the nucleotide level but is distinct from Hox-2.1 and Hox-2.2, also maps to this region of human chromosome 17 and is probably another member of the Hox-2 cluster of homeo box-containing genes. The third cDNA clone, c8, in which the homeo box is approximately 84% homologous to the mouse Hox-1.1 homeo box region on mouse chromosome 6, maps to chromosome region 12q12----12q13, a region that is involved in chromosome abnormalities in human seminomas and teratomas. The fourth cDNA clone, c13, whose homeo box is approximately 73% homologous to the Hox-2.2 homeo box sequence, is located at chromosome region 2q31----q37. The human homeo box-containing cluster of genes at chromosome region 17q21 is the human cognate of the mouse homeo box-containing gene cluster on mouse chromosome 11. Other mouse homeo box-containing genes of the Antennapedia class (class I) map to mouse chromosomes 6 (Hox-1, proximal to the IgK locus) and 15 (Hox-3). A mouse gene, En-1, with an engrailed-like homeo box (class II) and flanking region maps to mouse chromosome 1 (near the dominant hemimelia gene). Neither of the class I homeo box-containing genes--c8 and c13--maps to a region of obvious homology to chromosomal positions of the presently known mouse homeo box-containing genes.     

Estrogen Induction of Cytochrome c Oxidase Subunit III in Rat Hippocampus

Differential screening of a cDNA library prepared from mRNA of the hippocampus of estrogen-stimulated ovariectomized female rats led to the identification of a single estrogen-induced clone. Analysis of the sequence identified this cDNA as the gene coding for subunit III of the enzyme cytochrome c oxidase. Cytochrome c oxidase subunit III mRNA levels significantly increased as early as 3 h following the administration of a single dose of hormone. This effect was visible in the hippocampus and in the hypothalamus, but not in the other brain areas examined. Because subunit III of the cytochrome c oxidase is of mitochondrial origin, the mechanism involved in the estrogenic effect is still unknown. The observation that the activity of cytochrome c oxidase can also be induced by estrogens in the hippocampus indicates that this induction may be secondary to the increased expression of the other subunits of cytochrome c oxidase or to the general increase of neuronal activity.     

Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.

The six small subunits (IV-VII, VIIa, VIII) of yeast cytochrome c oxidase are encoded by nuclear genes and imported into the mitochondria. We have isolated the gene for subunit IV from a yeast genomic clone bank and determined its complete nucleotide sequence. We have also isolated subunit IV from purified yeast cytochrome c oxidase and determined most of its amino acid sequence which confirms the positioning of approximately 90% of the amino acid residues. The sequence comparison shows that the coding sequence of the gene lacks introns and that subunit IV is made as a precursor with an amino-terminal extension of 25 residues, five of which are basic and none of them acidic. Precursor processing involves cleavage of a Leu-Gln bond.     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.