cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

The expression of a functional cDNA encoding the chicken cytochrome P-450arom (aromatase) that catalyzes the formation of estrogen from androgen

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.     

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.     

Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX

A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.     

Identification of a zeta-crystallin (quinone reductase)-like 1 gene (CRYZL1) mapped to human chromosome 21q22.1

To identify a new gene(s) located on the yeast artificial chromosome (YAC) clone D142H8 that was mapped to human chromosome 21q22.1, purified YAC DNA from the clone was utilized directly as a probe to screen a human brain cDNA library after the suppression of human repetitive DNA. One cDNA clone hybridizing specifically to the YAC D142H8 DNA was identified. The clone has an insert of 1341 bp and the longest open reading frame of 349 amino acids. A search of GenBank revealed that the clone has a high degree of homology to zeta-crystallin (quinone reductase) at the amino acid level, and its nucleotide sequence represents the expressed sequence from the 50-kb segment of the human chromosome 21q11.1. Thus a new gene was named CRYZL1 (zeta-crystalline-like 1). Genomic Southern blot with total human and yeast DNAs suggests that CRYZL1 might be a single-copy gene. The fluorescence in situ hybridization procedure was applied, and the results showed that the gene mapped to the human chromosome 21q22.1 subband. The CRYZL1 mRNA was expressed in heart, brain, skeletal muscle, kidney, pancreas, liver, and lungs but at different levels in different tissues.     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.     

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.     

Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C

cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini.     

Localization of the human prealbumin gene to chromosome 18

A human liver cDNA library was screened using an oligonucleotide probe based on the amino acid sequence of human prealbumin. The cDNA insert of one positive clone was sequenced and found to contain the entire coding sequence of human prealbumin plus untranslated 5' and 3' regions. This cDNA was used to probe DNA from a panel of mouse/human somatic cell hybrids. Only those hybrids containing human chromosome 18 showed the human-specific hybridization pattern, thereby localizing the human prealbumin gene to this chromosome.     

Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines.

Rat monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned. A cDNA library was constructed from mRNA extracted from Con A-stimulated rat spleen cells. After 2 rounds of screening with a radiolabeled oligonucleotide probe and DNA sequencing from plasmid DNAs, one clone which encoded for MCP-1 was obtained. The cDNA clone comprises 665 base pairs with an open reading frame which encodes for 148 amino acid protein. Spleen cells expressed a significant amount of MCP-1 mRNA without stimulus. But higher expression was observed when cells were stimulated with Con A. MCP-1 mRNA was also expressed in tumor cell lines, although the amount of expressed MCP-1 mRNA was different among cell lines.     

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.     

Purification, molecular cloning, and sequencing of salivary cystatin SA-1

A "long form" salivary thiol protease inhibitor, designated cystatin SA-I, was purified to homogeneity from human submandibular-sublingual saliva by sequential gel filtration and ion-exchange chromatography. Automated peptide sequencing data revealed that cystatin SA-I shares sequence homologies with salivary cystatin SN, except that it contains an additional octapeptide at its NH2 terminus. To further characterize the molecular basis of salivary cystatin diversity, a mixed-base oligonucleotide probe corresponding to a region within the NH2-terminal sequence of the salivary cystatins was synthesized. This probe was used to screen a portion of a human submandibular gland cDNA library. The cDNA insert of a clone, designated pBR HSMSF 10G5.1, carried the entire peptide coding sequence of cystatin SA-I. The secretory peptide signal coding sequence was immediately followed by a sequence encoding the eight amino acid residues found at the NH2 terminus of purified cystatin SA-I. To estimate the number of genes encoding cystatins in the human genome, fragments of the pBR HSMSF 10G5.1 insert were used as probes in Southern blot analyses of human genomic DNA. These analyses revealed that the human genome carries 4-7 homologous cystatin genes. Collectively, our data suggest that some of the diversity in salivary cystatins could be generated by expression of different members of a multigene family and by posttranslational proteolytic cleavage of NH2-terminal regions (cystatin SA-I to cystatin SN).     

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum.

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.