Existence of glucose-6-phosphate dehydrogenase-like locus on chromosome 17.

Hybridization of DNA samples prepared from flow-sorted human chromosomes with a cDNA probe for the X-linked glucose-6-phosphate dehydrogenase (G6PD) suggested the existence of the G6PD-like locus on chromosome 17. Southern hybridization analysis of endonuclease-digested DNA samples from the human-mouse hybrid cell line with human chromosome 17, and from control human and mouse cells, proved that not only X chromosomes, but also chromosome 17, contain DNA sequences that are hybridizable with the G6PD cDNA probe. The G6PD-like locus on chromosome 17 could be a putative pseudogene or a functional gene for the fetal brain-specific G6PD isozyme or other protein.     

Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris.

We sequenced the NH2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides.     

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Isolation and partial characterization of the gene for cytochrome P-450 3a (P-450ALC) and a second closely related gene

Two genes that hybridize to the cDNA for alcohol-inducible cytochrome P-450 form 3a (P-450ALC) have been isolated from a rabbit genomic library and characterized by restriction mapping, hybridization, and partial sequence analysis. The genes show extensive sequence similarity as judged by hybridization at high stringency to the coding region of P-450 3a cDNA. However, only gene 1 hybridizes under these conditions to the 3' nontranslated segment of P-450 3a cDNA. The hybridizing fragments derived from both cloned genes were found to be present in the genome of all rabbits examined by Southern blot analysis, indicating that the genes represent separate loci and are not polymorphic alleles. Partial sequence analysis indicated that gene 1 encodes P-450 3a. Gene 2, if transcribed, would encode a protein with greater than 96% sequence identity with P-450 3a in the NH2-terminal region.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Localization of the mouse Mcf-2 (Dbl) protooncogene within a conserved linkage group on the mouse X chromosome

A mouse cDNA probe homologous to the human MCF2 transforming sequence has been identified and partially cloned, and is used here to localize the gene on the mouse X chromosome. The human gene has been physically mapped to within 60 kb of the gene for coagulation factor IX, within a large conserved linkage group between the mouse and human genomes which extends from HPRT to G6PD on the X chromosomes of both mammalian species. In situ hybridization of the mouse Mcf-2 probe onto mouse metaphase chromosomes indicates that this gene lies in the same region of the X chromosome as Cf-9, the mouse gene for coagulation factor IX. Moreover, segregation of species-specific genomic DNA polymorphisms for Mcf-2 and Cf-9 in a total of 203 individuals derived from two large interspecific mouse backcross populations (which are also segregating for 17 other X-linked molecular markers) demonstrates that the mouse genes are separated by only 0.5 +/- 0.5 cM. Despite this short distance we were able to order Mcf-2 and Cf-9 relative to one another and other genes in this region. The mouse gene order Hprt-Cf-9-Mcf-2-G6pd predicts a similar ordering of genes on the human X chromosome, a gene order which has only recently been demonstrated by physical mapping. Thus, the map location and linkage relationships of the Mcf-2 gene are similar in man and mouse, and this unique protooncogenic locus is part of a conserved linkage group on the mammalian X chromosome.     

Genomic organization of glycinin genes in soybean

Glycinin is the predominant seed storage protein in most soybean varieties. Previously, five major genes (designated Gy1 to Gy5) encoding glycinin subunits have been described. In this report two new genes are identified and mapped: a glycinin pseudogene, gy6, and a functional gene, Gy7. Messenger RNA for the gy6 pseudogene is not detected in developing seeds. While Gy7 mRNA was present at the midmaturation stage of seed development in the soybean variety Resnik, the steady state amount of this message was at least an order of magnitude less-prevalent than the mRNA encoding each of the other five glycinin subunits. Even though the amino-acid sequence of the glycinin subunit G7 is related to the other five soybean 11S subunits, it does not fit into either the Group-1 (G1, G2, G3) or the Group-2 (G4, G5) glycinin subunit families. The Gy7 gene is tandemly linked 3' to Gy3 on Linkage Group L (chromosome 19) of the public molecular linkage map. By contrast, the gy6 gene occupies a locus downstream from Gy2 on Linkage Group N (chromosome 3) in a region that is related to the position where Gy7 is located on chromosome 19.     

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

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.     

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH₂-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH₂-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Assignment of human genes for phosphorylase kinase subunits alpha (PHKA) to Xq12-q13 and beta (PHKB) to 16q12-q13.

Phosphorylase kinase (PHK), the enzyme that activates glycogen phosphorylases in muscle, liver, and other tissues, is composed of four different subunits. Recently isolated rabbit muscle cDNAs for the larger two subunits, alpha and beta, have been used to map the location of their cognate sequences on human chromosomes. Southern blot analysis of rodent x human somatic cell hybrid panels, as well as in situ chromosomal hybridization, have provided evidence of single sites for both genes. The alpha subunit gene (PHKA) is located on the proximal long arm of the X chromosome in region Xq12-q13 near the locus for phosphoglycerate kinase (PGK1). X-linked mutations leading to PHK deficiency, known to exist in humans and mice, are likely to involve this locus. This hypothesis is consistent with the proximity of the Phk and Pgk-1 loci on the mouse X chromosome. In contrast, the beta subunit gene (PHKB) was found to be autosomal and was mapped to chromosome 16, region q12-q13 on the proximal long arm. Several different autosomally inherited forms of PHK deficiency for which the PHKB could be a candidate gene have been described in humans and rats.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Isolation of human glucose-6-phosphate dehydrogenase (G6PD) cDNA clones: primary structure of the protein and unusual 5'' non-coding region.

Glucose-6-phosphate dehydrogenase (G6PD) is an ubiquitous enzyme which by determining the NADPH level has a crucial role in NADPH-mediated reductive processes in all cells (1). The structural gene for G6PD, Gd, is X-linked in mammals and on the basis of its expression in many tissues, it can be regarded as a typical "housekeeping" gene (2). Over 300 variants of the protein are known, many of which have deficient enzyme activity. Nearly 100 of these variants are polymorphic in various populations (3). The mammalian enzyme is a homodimer or a homotetramer with a subunit molecular weight of approximately 56000 daltons (4). Here we report the isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines. These clones have enabled us to sequence the entire coding region of Gd. Thus, the entire amino acid sequence of human G6PD is provided for the first time. This work is the first step for structural analysis of G6PD variants and for an understanding of the biological features of this enzyme at the molecular level.     

Structure and heterologous expression of a gene encoding fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

A full-length cDNA clone encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana (AtF2KP) was isolated. The encoded protein is composed of two different regions: (i) a 400 amino acid COOH-terminal region, covering the catalytic region of the protein which is homologous to enzymes from other eukaryotes. This region is highly conserved among plant species (88% identity to spinach F2KP). (ii) A 345 amino acid plant-specific NH(2)-terminal region, with 59% identity to spinach F2KP, which is composed of homologous motifs and intermittent variable sequences. Western blots show that F2KP from several plant species migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a similar sized (93 kDa) protein. AtF2KP was expressed in Escherichia coli as a full length and a truncated (without the NH(2)-terminal region) fusion protein. Both forms had kinase as well as phosphatase activity, but presence of the NH(2)-terminal region influenced the ratio between the two activities. It is suggested that the NH(2)-terminal region represents a regulatory region, which defines specific properties of the plant enzymes. A genomic clone for the corresponding gene, AtF2KP, was isolated. The clone (9519 bp) included 23 exons, 22 introns and the promoter sequence. Southern blot analysis showed only one copy of the gene in the A. thaliana genome.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.