Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase.

We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase θ. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase-helicase involved in repair of interstrand crosslinks. The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31.

cDNA cloning and genomic organization of enhancer of split groucho gene from nematode Caenorhabditis elegans.

This first genomic Enhancer of split groucho (ESG) gene and its full length complementary DNA (cDNA) from nematode C. elegans were cloned and sequenced via homology with the corresponding Drosophila groucho cDNA. The cDNA of 2.1-Kb encodes a protein of 612 amino acids, and the nematode ESG protein is the smallest and most different in structure compared to all ESG related proteins. The gene isolated is 4,246-bp in size, including 1,219-bp promoter region. A putative TATA-box at position -1166, two consensus sequence of ACTGG, characteristic of leader binding protein-1 (LBP-1) binding motifs at position -563 and -211 and nine CAAT boxes were found in the promoter region of ESG gene. The protein-coding sequence is interrupted by five introns. The length of introns 1 to 5 is 52, 252, 87, 53 and 518 bp, respectively. The overall structural relationships of the ESG-related proteins among human, mouse, rat, Xenopus, Drosophila and nematode were also analyzed.

Expression of human prostatic acid phosphatase and prostate specific antigen genes in neoplastic and benign tissues.

Expression of human prostatic acid phosphatase (ACPP) and prostate specific antigen (PSA) genes in prostatic carcinoma (CAP) and benign prostatic hyperplasia (BPH) was investigated by northern blot analyses. The expressions of ACPP and PSA, as well as the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase-muscle (LDH-A), were elevated significantly in prostatic carcinoma when compared with the expressions of these genes in benign prostatic hyperplasia in the same patient. The expression of the actin gene in both neoplastic and benign hyperplasia remained the same.

Nucleotide sequence of human prostatic acid phosphatase ACPP gene, including seven Alu repeats.

The protein-coding sequence of the human ACPP gene was shown to be interrupted by nine introns. The length of intron 1 was at least 5kb, while the sizes of introns 2 to 9 were estimated to be 3.4Kb, 0.5Kb, 5.5Kb, 5.0Kb, 2.0Kb, 5.0Kb, 2.5Kb and 3.5Kb, respectively. Therefore, the human ACPP gene has the size of more than 40kb. Thus far, the genomic sequence of 16,273 nucleotides, including the putative promoter region and seven Alu-repeats, have been determined. Three of these Alu-repeats are located immediately upstream to exon 1 and another is identified in intron 1. All of these Alu-sequences exhibit more than 85% identity to the consensus Alu-sequence.

The prostatic acid phosphatase (ACPP) gene is localized to human chromosome 3q21-q23.

Human prostatic acid phosphatase (ACPP) has been used as a diagnostic marker for prostate cancer. It is synthesized under androgen regulation and secreted by the epithelial cells of the prostate gland. We have confirmed the previous assignment of the ACPP gene to chromosome 3 by probing a panel of 25 human-Chinese hamster somatic cell hybrids, and we have further localized the ACPP gene to chromosome 3q21-q23 by fluorescence in situ hybridization.

Structure of human prostatic acid phosphatase gene.

Two cDNA clones containing the complete protein-coding sequence of 1,188 nucleotides as well as the 5' and 3' non-coding regions of human prostatic acid phosphatase (PAP) were isolated and sequenced. The size of PAP mRNAs from benign prostate hyperplasia and cancerous prostate was estimated to be 3.2Kb, indicating that the 3' downstream polyadenylation signal was used. Several genomic clones containing parts of the human PAP gene were isolated and the nucleotide sequence of ten exons and their flanking regions was determined. The protein-coding sequence of the human PAP gene was interrupted by nine introns. The positions of all nine introns present in the human PAP gene were homologous to those of the first nine introns in the human lysosomal acid phosphatase (LAP) gene. However, the last (11th) exon of the LAP gene encoding the COOH-terminal domain, which includes a transmembrane segment, was found to be absent in human PAP gene. Southern blot analysis of ten mammalian genomic DNAs gave multiple EcoRI fragments. The data of human genomic DNAs were consistent with the total length of the PAP gene of at least 50 kilobases.

The cDNA and protein sequences of mouse lactate dehydrogenase B. Molecular evolution of vertebrate lactate dehydrogenase genes A (muscle), B (heart) and C (testis).

Mouse lactate dehydrogenase-B cDNAs were isolated from cDNA libraries of macrophage (ICR strain) and thymus (F1 hybrid of C57BL/6 and CBA strains), and their nucleotide sequences determined. The lactate dehydrogenase-B cDNA insert of thymus clone mB188 consists of the protein-coding sequence (1002 nucleotides), the 5' (46 nucleotides) and 3' (190 nucleotides) non-coding regions, and poly(A) tail (19 nucleotides), while macrophage clone mB168 contains a partial lactate dehydrogenase cDNA insert from codon no. 55 to the poly(A) tail. Seven silent nucleotide substitutions at codon no. 142, 143, 186, 187, 241, 285 and 292, as well as a single nucleotide change in the 3' non-coding region, were found between these different strains of mice. The predicted sequence of 333 amino acids, excluding initiation methionine, was confirmed by sequencing and/or compositional analyses of a total of 103 (31%) amino acids from tryptic peptides of mouse lactate dehydrogenase-B protein. The nucleotide sequence of the mouse coding region for lactate dehydrogenase B shows 86% identity with that of the human isoenzyme, and only eight of the 139 nucleotide differences resulted in amino acid substitutions at residues 10, 13, 14, 17, 52, 132, 236 and 317. The rates of nucleotide substitutions at synonymous and nonsynonymous sites in the mammalian lactate dehydrogenase genes are calculated. The rates of synonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart) are considerably higher than the average rate computed from human and rodent genes. The rates of nonsynonymous substitutions for lactate dehydrogenase genes A (muscle) and B (heart), particularly the latter, are highly conservative. The rates of synonymous and nonsynonymous substitutions for the lactate dehydrogenase-C gene are about the same as the average rates for mammalian genes. A phylogenetic tree of vertebrate lactate dehydrogenase protein sequences is constructed. In agreement with the previous results, this analysis further indicates that lactate dehydrogenase-C gene branched off earlier than did lactate dehydrogenase-A and lactate dehydrogenase-B genes.

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase.

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.

Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human LDH-A, skeletal muscle isoenzyme.

Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human skeletal muscle isoenzyme, since the partial amino acid sequences of human liver LDH-K/A protein were found to be identical with the known primary structure of human LDH-A isoenzyme and the LDH-A isoenzymes from human placenta and bovine muscle were shown to be tyrosylphosphorylated. This tyrosylphosphorylated LDH-K/A protein was also found to be complexed with 21 kD, 30 kD, and 56 kD proteins.

Mapping of human lactate dehydrogenase-A, -B, and -C genes and their related sequences: the gene for LDHC is located with that for LDHA on chromosome 11.

Human testis-specific lactate dehydrogenase-C (LDHC) gene-related sequences are located with the LDHA gene on chromosome 11. The LDHB gene is on chromosome 12. Chromosomes 1, 2, 4, 9, and 10 appear to contain LDHA gene-related sequences, whereas the X chromosome and chromosome 13 possess LDHB gene-related sequences.

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].

Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse.

Mouse sperm-specific lactate dehydrogenase-C (LDH-C) cDNA was cloned and sequenced from lambda gt11 expression library. The LDH-C cDNA insert of 1236 bp consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (113 bp) non-coding regions, and the poly(A) tail (70 bp). The Northern blot analysis of poly(A)-containing RNAs from mouse testes and liver indicates that the LDH-C gene is expressed in testes but not in liver, and that its mRNA is approx. 1400 nucleotides in length. The nucleotide and amino acid sequences of the mouse LDH-C cDNA show 73% and 72% homologies, respectively, with those of the mouse LDH-A. The Southern blot analysis of genomic DNAs from mouse liver and human placenta indicates the presence of multiple LDH-C gene-related sequences.

Molecular nature of spontaneous mutations in mouse lactate dehydrogenase-A processed pseudogenes.

The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences to two different mouse lactate dehydrogenase-A processed pseudogenes, M11 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M11, the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22 nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M11 and M14, as well as that of pseudogene M10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.

Identification of the mouse low-salt-eluting single-stranded DNA-binding protein as a mammalian lactate dehydrogenase-A isoenzyme.

A 36,000-Mr protein purified from mouse myeloma on the basis of selective binding to a single-stranded DNA (ssDNA)-cellulose column has been identified as the lactate dehydrogenase A (LDH-A) subunit. A homogeneous preparation of this mouse myeloma ssDNA-binding protein, termed the 'low-salt-eluting protein', was found to possess LDH activity, and rabbit antiserum prepared against this protein was shown to cross-react with purified 36,000-Mr LDH-A subunits from mouse and bovine sources. In addition, bovine and human LHD-A4 isoenzymes were shown to be capable of binding ssDNA. These enzymic and immunological identities with LDH-A were not observed with purified helix-destabilizing protein 1 from mouse myeloma. A model for ssDNA-LDH binding is discussed.

Genomic organization of human lactate dehydrogenase-A gene.

A human genomic clone containing the lactate dehydrogenase-A (LDH-A) gene of approx. 12 kilobases in length was isolated and characterized. The protein-coding sequence is interrupted by six introns, and the positions of these introns are at the random coil regions or near the ends of secondary structures located on the surface of the LDH-A molecule. An additional intron is present at 24 nucleotides 5' to the translation initiation codon ATG, while the 3' untranslated sequence of 565 nucleotides is not interrupted. The genomic blot analysis of human placenta DNA indicates the presence of multiple LDH-A gene-related sequences.

Protein structure and gene organization of mouse lactate dehydrogenase-A isozyme.

The complete covalent structure of the 331 amino acids of mouse lactate dehydrogenase (LDH) A4 isozyme has been determined by sequence analyses of both the protein and the genomic DNA. The mouse LDH-A gene spans a length of at least 7000 bases from the translation initiation codon ATG to the end of the 3' untranslated region, and it contains six introns that interrupt the protein-coding sequence. The relationships between the exon-intron organization of LDH-A gene and the structural-functional domains of the protein are discussed.

Glycoprotein exhibiting immunological and enzymatic activities of human prostatic acid phosphatase.

A glycoprotein (GP) which is immunochemically and biologically related to human prostatic acid phosphatase (PAP) has been isolated from human seminal plasma by ammonium sulfate precipitation followed by sequential concanavalin A:Sepharose 4B column, anion-exchange chromatography and gel filtration. The purified GP was shown to be homogeneous by disc- and sodium dodecyl sulfate: polyacrylamide gel electrophoresis. The apparent molecular weight of purified GP was estimated to be 50,000 by gel filtration and 45,000 by sodium dodecyl sulfate: gel electrophoresis. In gel diffusion against antiserum to purified PAP, a partial immunological identity was shown between GP and PAP. This was further confirmed by an inhibition reaction between GP and antiserum to purified GP by PAP. Significantly, 30% of PAP enzyme activity was inhibited by anti-GP antiserum, while only 5% was inhibited by anti-PAP antiserum. Purified GP was shown to exhibit a weak, but significant, acid phosphatase activity by hydrolyzing alpha-naphthyl phosphate at pH 5.6. The Km and Vmax for GP are 1.6 X 10(-4) M and 0.056 mumol/min/microgram protein, respectively, using alpha-naphthyl phosphate as the substrate. In the presence of anti-PAP antibody, the enzyme activity of GP was enhanced severalfold. Furthermore, the acid phosphatase activity of GP also was inhibited by tartrate, which is the most commonly used inhibitor for PAP. GP and PAP were found to have different carbohydrate content, amino acid composition, amino-terminal sequence, and peptide map. Thus, GP represents a newly identified protein. The significance of these results at molecular and clinical levels is discussed.

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes.

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.

Evolutionary relationships of vertebrate lactate dehydrogenase isozymes A4 (muscle), B4 (heart), and C4 (testis).

The amino acid sequence variation among seven lactate dehydrogenase isozymes from dogfish muscle, chicken muscle and heart, pig muscle and heart, and mouse and rat testes were compared with respect to the whole lactate dehydrogenase polypeptide chain as well as their four functional domains. The coenzyme-binding domain is more conserved than the substrate-binding domain. The sequence of the loop and helix alpha D region of testicular LDH-C4 isozymes is very different from those of somatic LDH-A4 and LDH-B4 isozymes, while the NH2-terminal arm is extremely variable. The most parsimonious phylogenetic tree among these seven vertebrate lactate dehydrogenase sequences clearly indicates that the LDH-A4 and LDH-B4 isozymes are more closely related to each other than either to the LDH-C4 isozymes.

Amino acid sequence of small and large subunits of seed storage protein from Ricinus communis.

The low molecular weight, glutamine-rich storage protein isolated from the seeds of Ricinus communis (castor beans) has been shown to consist of two different polypeptide chains linked by disulfide bond(s). The small subunit is composed of 34 amino acids with a proline at its NH2 terminus, whereas the large subunit contains 61 amino acids with a cyclized glutamine as the NH2-terminal residue. The complete amino acid sequence of both subunits has been determined through characterization of the isolated subunits and selected peptides from trypsin, chymotrypsin, thermolysin, and cyanogen bromide cleavage. The intact protein possesses a large number of glutaminyl and half-cystinyl residues and exhibits sequence heterogeneity as observed from peptide sequences. Comparison of the sequence of this protein and those of other seed proteins indicates some structural similarities between them. The amino acid sequences of the two polypeptide chains of castor bean storage protein are: (formula, see text).