Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.     

Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.     

Molecular cloning and characterization of the human NIMA-related protein kinase 3 gene (NEK3)

NEKs (NIMA-related kinases) are a group of protein kinases sharing high amino acid sequence identities with NIMA (never in mitosis gene a) which control mitosis in Aspergillus nidulans. We have cloned a cDNA for human NEK3, a novel human gene structurally related to NIMA, by RT-PCR. Its open reading frame encodes a protein of 489 amino acid residues with the calculated molecular mass of 56.0 kDa and a predicted pI of 6.58. Phylogenetic analysis suggests that mouse and human NEK3s constitute a subfamily within the NIMA family of protein kinases. The expression pattern of NEK3 was studied by RT-PCR and a high level of expression was detected in testis, ovary, and brain, with low-level expression being detected in most of the tissues studied. NEK3 mRNA was detected in all the proliferating cell lines studied, and the amount did not change during the cell cycle. The human NEK3 gene was assigned to human chromosome 13 by somatic cell hybrids and 13q14.2 by radiation hybrid mapping.     

Rat ribophorin II: molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum.

We report here the complete nucleotide sequence of rat ribophorin II. The predicted amino acid sequence is highly homologous to the corresponding human protein and consists of 631 amino acid residues, including a 22 amino acid N-terminal cleavable signal sequence, and a single 23 amino acid putative transmembrane domain. Northern blot analysis reveals a single -2.4 kb message expressed in a number of rat cell lines and in adult liver. The gene was mapped to mouse chromosome 2, close to the Src proto-oncogene.     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.     

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa

The BAZF gene has recently been identified as a novel homologue of the BCL6 oncogene. Here we cloned the human BAZF gene using murine BAZF as a probe. The predicted amino acid sequence was 91% identical to that of murine BAZF. The BTB/POZ and zinc finger domains were almost completely conserved between human and murine BAZF. Fluorescence in situ hybridization analysis revealed that the human BAZF gene is located on chromosome 17p13.1. Although expression of human BAZF mRNA was ubiquitously detected in human tissues, abundant expression was detected in heart and placenta. BAZF mRNA was expressed in some immature B cell lines and erythroleukemia cell lines. The expression in a human erythroleukemia cell line, HEL cells, was upregulated during megakaryocytic differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate. These expression patterns of BAZF mRNA suggest that BAZF may regulate differentiation in stages or lineages that are different from those regulated by BCL6.     

Cloning and characterization of a new functional human aldehyde dehydrogenase gene

We cloned a new functional ALDH gene (ALDHx) from a human genomic library in cosmid pWE-15 by screening with a 29-nucleotide probe partially matched to a conserved region of the ALDH1 and ALDH2 genes. The new ALDHx gene does not contain introns in the coding sequence for 517 amino acid residues. The degree of resemblance between the deduced amino acid sequences of the new ALDHx gene and the ALDH2 gene is 72.5% (alignment of 517 amino acid residues), while that between the ALDHx and the ALDH1 gene is 64.6% (alignment of 500 amino acid residues). The amino acid residues (Cys-162, Cys-302, Glu-268, Glu-487, Gly-223, Gly-225, Gly-229, Gly-245 and Gly-250), which exist in both ALDH1 and ALDH2 isozymes and have been implicated in functional and structural importance, are also preserved in the deduced sequence of the new ALDHx gene. Northern blot hybridization with ALDHx probe revealed the existence of a unique mRNA band (3.0 kilobases) in the human liver and testis tissues. Using the new ALDHx probe, we cloned the cDNA of the gene from a human testis cDNA library in lambda gt11 vector. The nucleotide sequence of the cDNA differs from that of the genomic sequence at three nucleotide positions resulting in the exchange of 2 deduced amino acid residues. These positions are polymorphic as further demonstrated by the PCR amplification of the targeted region followed by nucleotide sequence analysis of the genomic DNA from eight unrelated individuals. Alignment of the genomic and cDNA sequence indicates that although the ALDHx gene appears to have no intron in its coding sequence, an intron of 2.6 kilobases is found to interrupt the 5'-untranslated (5'-UT) sequence. Primary extension and S1 mapping analysis indicate the existence of at least two 5'-UT exons. The new ALDHx gene was assigned to chromosome 9 by Southern blot hybridization of DNA samples from a panel of rodent-human hybrid cell lines.     

Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.     

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.     

Cloning of murine cysteine sulfinic acid decarboxylase and its mRNA expression in murine tissues

Cysteine sulfinic acid decarboxylase (CSD) is the rate-limiting enzyme for biosynthesis of taurine which is essential to biological processes such as development of the brain and eye, reproduction, osmoregulation as well as the anti-inflammatory activity of leukocytes. We report the cDNA sequence of murine CSD that predicts a polypeptide of 493 amino acids. This protein shares 98% and 90% of amino acids with rat and human CSD, respectively, indicating that it is a true ortholog of CSD. Northern blot analysis revealed that CSD mRNA is expressed in kidney and liver, and was not detected in lymphoid tissues and lung. The nucleotide sequence of murine CSD should be useful for genetic manipulation of the CSD gene.     

Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells

Herein we describe the isolation of a cDNA encoding a novel human Toll-like receptor (hTLR) that we term hTLR10. Human TLR10 contains 811 amino acid residues. Deduced amino acid sequence analysis reveals that like the other known hTLRs (hTLR1-9) it is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor. Phylogenetic analysis indicates that among all the hTLRs, hTLR10 is most closely related to hTLR1 and hTLR6; the overall amino acid identity is 50% and 49%, respectively. hTLR10 mRNA is most highly expressed in lymphoid tissues such as spleen, lymph node, thymus, and tonsil. Expression analysis of cell lines indicates a predominance in a variety of immune cell types. Thus, hTLR10 is preferentially expressed in tissues and cells involved in immune responses.     

A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues

We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.