Mono(ADP-ribosyl)ation of 2'-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly.

Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A small middle dotB structure-function organization like cholera or diphtheria toxin, where the "A" domain (N-terminal) exhibits ADP-ribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of beta-[adenylate-(32)P]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 110 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG small middle dotdC, but not dA small middle dotdT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N(2) of 2'-deoxyguanosine to yield N(2)-(alpha-ADP-ribos-1-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including (1)H- and (13)C-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N(2)-(D-ribos-1-yl)-2'-deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the (32)P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2'-deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation.

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

Purification and cloning of pierisin-2, an apoptosis-inducing protein from the cabbage butterfly, Pieris brassicae.

The cabbage butterfly Pieris rapae contains a strong apoptosis-inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP-ribosylation. Here we report the purification and cloning of an apoptosis-inducing substance, designated as pierisin-2, from another cabbage butterfly, Pieris brassicae. Pierisin-2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis-inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin-1, from P. rapae. cDNA cloning of pierisin-2 was performed on the basis of the partial amino-acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850-amino-acid protein with a calculated molecular mass of 97 986. The deduced amino-acid sequence of pierisin-2 was 91% identical with that of pierisin-1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis-inducing activities against human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, similar to the purified native pierisin-2 from the pupae. Pierisin-2 shows regional sequence similarities with certain ADP-ribosylating toxins such as the A-subunit of cholera toxin. The results from site-directed mutagenesis at Glu165, a conserved residue among ADP-ribosylating enzymes necessary for NAD binding, and from experiments with ADP-ribosylating enzyme inhibitors suggested that pierisin-2 could be considered as an ADP-ribosylating toxin like pierisin-1.

Suppression by flavonoids of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells: structure-activity relationship.

Cyclooxygenase-2 (COX-2) plays an important role in carcinogenesis. Investigation of the suppressive action of twelve flavonoids of different chemical classes on the transcriptional activity of the COX-2 gene in human colon cancer DLD-1 cells using a reporter gene assay have revealed quercetin to be the most potent suppressor of COX-2 transcription (IC50 = 10.5 microM), while catechin and epicatechin showed weak activity (IC50 = 415.3 microM). Flavonoids have three heterocyclic rings as a common structure. A structure-activity study indicated that the number of hydroxyl groups on the B ring and an oxo group at the 4-position of the C ring are important in the suppression of COX-2 transcriptional activity. A low electron density of the oxygen atom in the hydroxyl group of the A ring was also important. Further examination of the role of the hydroxyl group in the A ring showed that bromination of resacetophenone to give 3,5-dibromo-2,4-dihydroxyacetophenone resulted in a 6.8-fold increase in potency for suppressing COX-2 promoter activity. These results provide a basis for the design of improved suppressors of COX-2 transcriptional activity.

Inhibition by parthenolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1.

Excessive nitric oxide production by inducible nitric oxide synthase (iNOS) in stimulated inflammatory cells is thought to be a causative factor of cellular injury in inflammatory disease states. Compounds inhibiting iNOS transcriptional activity in inflammatory cells are potentially anti-inflammatory. An assay method for estimating iNOS transcriptional activity in the human monocyte cell line THP-1 was established using a luciferase reporter gene system. In this study, we demonstrate that parthenolide, the predominant sesquiterpene lactone in European feverfew (Tanacetum parthenium), exerts potent inhibitory effects on the promoter activity of the iNOS gene in THP-1 cells. Parthenolide effectively suppressed iNOS promoter activity in a dose-dependent manner at concentrations higher than 2. 5 microM, with an IC(50) of about 10 microM. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), significantly increased the iNOS promoter-dependent reporter gene activity, and the TPA-induced increase in iNOS promoter activity was effectively suppressed by parthenolide, with an IC(50) of approximately 2 microM. The present findings may further explain the anti-inflammatory property of parthenolide.

Suppression of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells by chemopreventive agents with a resorcin-type structure.

Cyclooxygenase-2 (COX-2) is abundantly expressed in colon cancer cells. It has been reported that inhibition of COX-2 enzyme activity is shown to prevent colon carcinogenesis. Thus, suppression of COX-2 expression may also be an effective chemopreventive strategy. In the present study, we constructed a beta-galactosidase reporter gene system in human colon cancer DLD-1 cells, and measured COX-2 promoter-dependent transcriptional activity in the cells. Interferon gamma suppressed this COX-2 promoter activity, while 12-O-tetradecanoylphorbol-13-acetate and transforming growth factor alpha (TGFalpha) exerted enhancing effects. We then tested the influence of 14 candidate cancer chemopreventive compounds on COX-2 promoter activity. Chemopreventive agents such as quercetin, kaempferol, genistein, resveratrol and resorcinol, all having a common resorcin moiety, were found to effectively suppress the COX-2 promoter activity with and without TGFalpha-stimulation in DLD-1 cells. Since all these compounds have a resorcin moiety as a common structure, a resorcin-type structure may play an active role in the inhibition of COX-2 expression in colon cancer cells.

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: possible involvement of ADP-ribosylation in its activity.

We have previously reported that the cabbage butterfly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS/PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADP-ribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisin-induced apoptosis. These results suggest that the apoptosis-inducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.

Inhibition by berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells.

The enzyme cyclooxygenase-2 (COX-2) is abundantly expressed in colon cancer cells and plays a key role in colon tumorigenesis. Compounds inhibiting COX-2 transcriptional activity have therefore potentially a chemopreventive property against colon tumor formation. An assay method for estimating COX-2 transcriptional activity in human colon cancer cells was established using a beta-galactosidase reporter gene system, and examination was made of various medicinal herbs and their ingredients for an inhibitory effect on COX-2 transcriptional activity. We found that berberine, an isoquinoline alkaloid present in plants of the genera Berberis and Coptis, effectively inhibits COX-2 transcriptional activity in colon cancer cells in a dose- and time-dependent manner at concentrations higher than 0.3 microM. The present findings may further explain the mechanism of anti-inflammatory and anti-tumor promoting effects of berberine.

Inhibition of activator protein 1 activity by berberine in human hepatoma cells.

Activator protein 1 (AP-1) is a transcription factor which plays a critical role in inflammation and carcinogenesis. The present study was conducted to investigate the effect of berberine, an isoquinoline alkaloid present in plants of the genera Berberis and Coptis, on the activity of AP-1 using a reporter gene assay in human hepatoma cells. Berberine was shown to inhibit AP-1 activity in a dose- and time-dependent manner at concentrations higher than 0.3 microM. Berberine inhibited AP-1 activity almost completely as low as 10 microM after 48 h treatment. The inhibitory effect on AP-1 activity in cancer cells may further explain the anti-tumor promoting activity of berberine.

Rat maf-related factors: the specificities of DNA binding and heterodimer formation.

maf is a family of genes encoding bZIP transcription factors. We isolated two cellular maf-related cDNAs, maf-1 (mafB) and maf-2 (c-maf), from rat and determined the specificities of DNA binding and heterodimer formation. Although both Mafs strongly bind to MARE, the consensus Maf recognition sequence (MARE, -TGCTGACTCAGCA-), originally identified by v-Maf protein Maf-1, recognizes a number of sequences containing only the first half of the MARE, -GCTGAC-. On the other hand, no such consensus short sequence could be determined for Maf-2. We determined the specificities of heterodimer formation with all members of the Jun and Fos family. In contrast to v-Maf which forms heterodimers with all Jun and Fos proteins, Maf-1 heterodimerizes with all four Fos proteins, but not at all with the three Jun proteins. Maf-2 heterodimerizes with c-Fos. We have also found that heterodimer formation of Maf-2 with c-Fos dramatically changes the specificity of DNA binding and trans-activation activity from that of the Maf-2 homodimer. These results show that Maf-1 and Maf-2 have significantly different properties and they might have different target genes and functions, in spite of the similarity of their bZip domain structure.

Differential expression of maf-1 and maf-2 genes in the developing rat lens.

PURPOSE: To examine the expression of maf-1 and maf-2 protocogenes in the developing rat lens. METHODS: Maf-1 and maf-2 transcripts were assayed in rat lenses on embryonic days 13 and 16 (E13 and E16) by in situ hybridization using single-stranded RNA probes. Proteins encoded by the maf-2 gene were assayed immunocytochemically in embryonic (E12, 13, 16, 19) and postnatal day 14 and 90 (P14 and P90) lenses. RESULTS: In embryonic lenses, we detected maf-1 messenger RNA (mRNA) in the lens epithelium and maf-2 mRNA diffusely distributed in the lens fiber cells. By immunocytochemistry, Maf-2 was detected on E12 in the nuclei of almost all lens pit cells. On days E13, E16, and E19, however, lens epithelial cells showed no immunoreactivity, but nuclei of fiber cells reacted strongly. On P14, nuclei containing Maf-2 protein were confined to the equator of the lens, but at 3 months of age, no Maf-2 could be detected in the rat lens. Western blotting showed that the anti-Maf-2 antiserum reacted with a single protein, of molecular weight approximately 39 kDa, in rat lens. CONCLUSIONS: Results showed the spatial and temporal regulation of maf gene expression and suggest that these genes participate in transcriptional regulation during the development of the lens in the rat.

[Study of rat maf-related gene products: recognition DNA sequences and heterodimer-formation].

The v-maf oncogene, originally identified as the transforming gene of the avian retrovirus AS42, encodes a protein with a basic-leucine zipper (bZIP) structure which is a typical motif for protein dimerization and DNA binding. The v-Maf protein forms homodimers and sometimes heterodimers with some bZIP proteins and works as a transcription factor. Recent studies of tissue-specific expression of cellular Maf family proteins suggest that Maf-related proteins play important roles in early development and cell differentiation. In our previous studies, two maf-related cDNA clones, maf-1 and maf-2, have been isolated from a rat liver cDNA library which facilitates the investigation of physiological roles of Mafs as well as their target genes in mammals. We determine here the binding DNA sequences of Maf-1 and Maf-2, respectively. Maf-1 recognizes a number of sequences containing a short consensus sequence, -GCTGAC-, half of the Maf recognition element (MARE) which has been previously identified for V-Maf. On the other hand, binding sequences of Maf-2 are limited and Maf-2 binds to the MARE preferentially. It is shown that dimer-forming specificities of Maf-1 and Maf-2 to Jun or Fos family proteins are variable. Maf-1 efficiently forms heterodimers with Fos family proteins. Compared to the case of Maf-2, though the DNA-binding specificity of the heterodimer depends on the kind of counterpart binding to Maf-1, a wide variety of target sequences are available for Maf-1. In contrast, Maf-2 shows restricted heterodimer-forming specificity and DNA-binding specificity, so that the target genes for Maf-2 are considered to be much more limited.

Suppression of rat glutathione transferase P expression by peroxisome proliferators: interaction between Jun and peroxisome proliferator-activated receptor alpha.

Glutathione transferase-P (GST-P) in rats is specifically expressed in precancerous lesions and in hepatomas induced by carcinogens or spontaneously arising hepatomas. GST-P expression in preneoplastic lesions is suppressed by peroxisome proliferators. To determine the mechanism of suppression of GST-P expression by peroxisome proliferators on a molecular level, we have analyzed the effects of peroxisome proliferators and their receptor (peroxisome proliferator-activated receptor alpha, PPAR alpha) on GST-P expression. GST-P gene expression linked to a reporter gene was specifically suppressed by cotransfection with a PPAR alpha expression plasmid in the presence of the peroxisome proliferator, clofibrate. The target element of the suppression was a 12-O-tetradecanoylphorbol-13-acetate-responsive element located 61 nucleotides upstream from the cap site, which is also internal to a Maf consensus binding sequence. Both Jun and Maf bind to this element and activate the gene having this element, but only Jun-activated expression was specifically inhibited by PPAR alpha. Expression of a transfected reporter gene linked to a PPAR-responsive element was inhibited by cotransfection with a Jun expression plasmid. These results suggest that PPAR alpha and Jun interact and share inhibitory activities, similar to Jun and the glucocorticoid receptor.

The C-terminal region, Arg201-Gln209, of glutathione S-transferase P contributes to stability of the active-site conformation.

The C-terminal region of rat glutathione S-transferase P (GST-P) was deleted by either carboxypeptidase (CPase) A and B or site-specific truncation to evaluate the role of the region in the catalytic mechanism. The C-terminal sequence from the 201st to 209th amino-acid residues is Arg-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln. When seven of the C-terminal amino-acid residues from the C-terminus were removed by the CPases, the catalytic activity decreased in parallel with the amino-acid removal, amounting to less than 5% of that of the wild-type GST-P. On the other hand, a decrease of the catalytic activity was observed in a different manner when the C-terminal sequence was site-specifically truncated. The VmaxGSH/KmGSH values of the mutants withthree (GSTd207-209), four (GSTd206-209) and seven (GSTd203-209) C-terminal amino-acid residues deleted, were comparable or similar to that of the wild-type GST-P, whereas those of five (GSTd205-209), six (GSTd204-209), and eight (GSTd202-209) amino-acid residue-truncated mutants decreased to 43%, 40%, and 19% of that of the wild-type GST-P, respectively. Similar results were obtained as for VmaxCDNB/KmCDNB. The nine amino-acid residue-truncated mutant showed no catalytic activity. Heat treatment at 50 degrees C for 5 min had little effect on the catalytic activities of the wild-type GST-P and GSTd204-209, whereas those of GSTd207-209, GSTd206-209, GSTd203-209 and GSTd202-209 decreased to 22%, 27%, 18% and 10%, respectively, compared to the catalytic activity of the non-treated enzymes. Considering these results, it is concluded that the C-terminal region, Arg201-Gln209, has an important role in stabilizing the active-site conformation.

Glutathione binding rat liver 13k protein is the homologue of the macrophage migration inhibitory factor.

We have cloned the cDNA of the rat liver 13k protein, that has glutathione binding activity, from a rat liver cDNA library. The nucleotide sequence of this cDNA predicts a protein of 115 amino acids. This protein has 99.1%, 89.6% and 73.0% homology with mouse, human and chicken macrophage migration inhibitory factor (MIF), respectively. This indicates that the rat liver 13k protein is homologue of the MIF. The N-terminal 25 amino acids show 35% sequence similarity with that of the rat glutathione transferase Yb subunit. Although the remaining C-terminal sequence has no sequence identity with glutathione transferase Yb subunit, about 50% homology was found, if conservative substitutions and gaps were taken into account. Northern blot analysis indicates that this gene is expressed in a wide variety of organs including brain, spleen, liver, muscle and kidney. Southern blot analysis suggests that the MIF gene has many pseudo-genes or/and closely related genes.

Haplotype diversity in the human red and green opsin genes: evidence for frequent sequence exchange in exon 3.

We studied polymorphisms in the coding sequences of the human red and green opsin genes of 133 Caucasian males. Eleven polymorphic sites were discovered in the red opsin gene, seven of which were in exon 3, three in exon 4 and one in exon 5. Polymorphisms at 8 of these sites resulted in amino acid substitutions which generated a total of 18 unique red opsins in the population. The substitutions at three (S180A, I230T, and A233S) of the 8 sites involve hydroxyl-bearing to non-polar amino acid residues, and are therefore likely to alter spectral characteristics of the red pigment. Eight polymorphic sites were observed in the green opsin coding sequences, six of which were in exon 3, one in exon 2 and one in exon 5. Five of the eight involved amino acid substitutions which generated 15 unique green opsins in the population. Substitutions at two of these sites involve hydroxyl-bearing vs. non-polar residues. Six polymorphisms, all of which are located in exon 3, are shared between the red and green opsin genes, essentially making it difficult to assign this exon to either of these genes. Markers in exon 3 are in partial linkage disequilibrium with those in exons 4 and 5, whereas the latter two are in strong linkage disequilibrium with each other. Furthermore, markers in the 5' region of exon 3 are also in only partial (54%) disequilibrium with those in the 3' region. The above results strongly suggest a history of frequent gene conversion, mainly localized to exon 3, in the lineages leading to the human red and green opsin genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotype-phenotype relationships in human red/green color-vision defects: molecular and psychophysical studies.

The relationship between the molecular structure of the X-linked red and green visual pigment genes and color-vision phenotype as ascertained by anomaloscopy was studied in 64 color-defective males. The great majority of red-green defects were associated with either the deletion of the green-pigment gene or the formation of 5' red-green hybrid genes or 5' green-red hybrid genes. A rapid PCR-based method allowed detection of hybrid genes, including those undetectable by Southern blot analysis, as well as more precise localization of the fusion points in hybrid genes. Protan color-vision defects appeared always associated with 5' red-green hybrid genes. Carriers of single red-green hybrid genes with fusion in introns 1-4 were protanopes. However, carriers of hybrid genes with red-green fusions in introns 2, 3, or 4 in the presence of additional normal green genes manifested as either protanopes or protanomalous trichromats, with the majority being protanomalous. Deutan defects were associated with green-pigment gene deletions, with 5' green-red hybrid genes, or, rarely, with 5' green-red-green hybrid genes. Complete green-pigment gene deletions or green-red fusions in intron 1 were usually associated with deuteranopia, although we unexpectedly found three carriers of a single red-pigment gene without any green-pigment genes to be deuteranomalous trichromats. All but one of the other deuteranomalous subjects had green-red hybrid genes with intron 1, 2, 3, or 4 fusions, as well as several normal green-pigment genes. The one exception had a grossly normal gene array, presumably with a more subtle mutation. Amino acid differences in exon 5 largely determine whether a hybrid gene will be more redlike or more greenlike in phenotype. Various discrepancies as to severity (dichromacy or trichromacy) remain unexplained but may arise because of variability of expression, postreceptoral variation, or both. When phenotypic color-vision defects exist, the kind of defect (protan or deutan) can be predicted by molecular analysis. Red-green hybrid genes are probably always associated with protan color-vision defects, while the presence of green-red hybrid genes may not always manifest phenotypically with color-vision defects. Four subjects who were found to have 5' green-red hybrid genes in addition to normal red- and green-pigment genes had normal color vision as determined by anomaloscopy. These were discovered among a group of 129 Caucasian males who had been recruited as volunteers for a vision study.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.

If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin.

A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E. coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E. coli at high efficiency, the soluble heme protein representing approximately 10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure-function analysis of horse heart myoglobin.

Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex.

Using an oligonucleotide probe designed on the basis of the N-terminal amino acid sequence of purified rat aldosterone synthase cytochrome P-450 [(1989) J. Biol. Chem. 264, 10935] we have isolated from rat adrenal cDNA library a 2687 base pair cDNA that encodes a protein of 500 amino acid residues. The deduced amino acid sequence contained the regions well conserved among all cytochrome P-450s sequenced to date, and also a portion (residues 25-44) which was identical to the N-terminal peptide sequence of rat aldosterone synthase cytochrome P-450. These results indicate that the cDNA encodes a precursor form of rat aldosterone synthase cytochrome P-450.