Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I

Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.     

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.     

Porcine granulin gene (GRN): molecular cloning, polymorphism and chromosomal localization.

GRN has been shown to have roles in multiple processes involved in cell growth, development and wound repair in rodents and humans. We have isolated the full-length cDNA of GRN gene encoding porcine granulin protein by in silico cloning, RT-PCR and RACE. The deduced amino acid indicated 71.5% identity with the corresponding human sequence and the seven and one-half granulins showed highly conservative between pig, human and murine. A single nucleotide substitution resulting in the amino acid change (ATG/Met --> TTG/Leu) was detected within exon 5. Allele frequencies in six pig breeds showed distinctive differences between those Chinese indigenous pig breeds and European pigs. Using the IMpRH panel, we mapped the porcine GRN gene to porcine chromosome 12p11-p13. Our data provide basic molecular information useful for the further investigation on the function of GRN gene.     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.     

Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase.

Murine macrophages have previously been shown to secrete a zinc-dependent proteinase that can degrade elastin. In this report, we identify murine macrophage elastase (MME) cDNA and show that it is a distinct member of the metalloproteinase gene family. Small amounts of MME were purified to homogeneity, and N-terminal amino acid sequence was obtained. This sequence was used to obtain a partial cDNA clone by the polymerase chain reaction; a cDNA library derived from a mouse macrophage-like cell line (P388D1) was screened with this probe. A full-length MME cDNA spanning approximately 1.8 kilobases contained an open reading frame of 1386 base pairs; the predicted molecular mass of the MME proenzyme is 53 kDa. The gene encoding MME is represented only once in the mouse genome and is located on chromosome 9. Despite a size that is similar to other metalloproteinases, MME is distinct, sharing only 33-48% amino acid homology with other metalloproteinases. In contrast to other metalloenzymes, MME appears to be rapidly processed to an active truncated form (N-terminal and C-terminal cleavage). We expressed recombinant MME in Escherichia coli and demonstrated that it has significant elastolytic activity that is specifically inhibited by the tissue inhibitor of metalloproteinases. MME is therefore a true metalloproteinase that may be involved in tissue injury and remodeling.     

Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution

Porcine organs and lung surfactant have medically important applications in both xenotransplantation and therapy. We have started to characterize porcine lung surfactant by cloning the cDNA of porcine surfactant protein D (SP-D). SP-D and SP-A are important mediators in innate immune defense for the lung and possibly other mucosal surfaces. Porcine SP-D will also be an important reagent for use in existing porcine animal models for human lung infections. The complete cDNA sequence of porcine SP-D, including the 5' and 3' untranslated regions, was determined from two overlapping bacteriophage clones and by PCR cloning. Three unique features were revealed from the porcine sequence in comparison to SP-D from other previously characterized species, making porcine SP-D an intriguing species addition to the SP-D/collectin family. The collagen region contains an extra cysteine residue, which may have important structural consequences. The other two differences, a potential glycosylation site and an insertion of three amino acids, lie in the loop regions of the carbohydrate recognition domain, close to the carbohydrate binding region and thus may have functional implications. These variations were ruled out as polymorphisms or mutations by confirming the sequence at the genomic level in four different pig breeds. Porcine SP-D was shown to localize primarily to the lung and with less abundance to the duodenum, jejunum, and ileum. The genes for SP-D and SP-A were also shown to colocalize to a region of porcine chromosome 14 that is syntenic with the human and murine collectin loci.     

Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Genetic recombination in Coprinus: III. Inlluence of gamma-irradiation and temperature treatments on meiotic recombination

Non-lethal doses of gamma-irradiation (5 krad) increased meiotic recombination in Coprinus lagopus when treatments were given at the beginning of karyogamy. The division stage at this time was judged to be late leptotene and the duration of the sensitive period was assessed to be 3–4 h. In C. lagopus the radiation-sensitive stage is distinct from the cold-sensitive stage (pachytene). The additive effect of irradiation at early karyogamy followed by cold treatment in pachytene suggested that the two factors influenced different steps in the recombination process. On the other hand, irradiation followed by heat treatment did not significantly alter recombination frequency as compared to single treatments. It was surmised that radiation and high temperature act on the same factor(s) or at the same steps to bring about a similar net result. It was suggested that irradiation at leptotene may cause single-strand breaks in DNA which eventually participate in exchange.     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.     

Seabream antiquitin: molecular cloning, tissue distribution, subcellular localization and functional expression

Subsequent to our earlier report on the first purification of antiquitin protein from seabream liver and demonstration of its enzymatic activity [FEBS Letters 516 (2002) 183-186], we report herein the cloning of its full-length cDNA sequence. The open reading frame encodes a protein of 511 amino acids. Results of RT-PCR indicate that antiquitin is highly expressed in both the seabream liver and kidney. Transfection studies in cultured eukaryotic cells provided further evidence that it is a cytosolic protein. Bacterial expression of the enzyme was also performed. The purified recombinant protein was demonstrated to exhibit similar kinetic properties as the native enzyme.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

The Drosophila ets-2 gene: molecular structure, chromosomal localization, and developmental expression

The cellular homologs of the ets gene from the avian erythroblastosis retrovirus E26 have been studied in chickens, humans, mice, and cats. In this report a further evolutionary step is taken by isolating and characterizing a Drosophila ets-related genomic clone. Sequence analysis of this clone has shown it to contain the 3' end of the v-ets gene, called ets-2, corresponding to the last two exons of chicken ets. The predicted amino acid sequence was found to have over 90% homology when compared to that of v-ets. This is the highest level of conservation observed for any previously characterized Drosophila oncogene homolog. Expression of the ets-2 gene occurs throughout development, but is highest during the embryonic and pupal stages. By in situ hybridization, the ets-2 chromosomal position was determined to be 58A/B which corresponds to no known phenotypic mutant. As this is a highly conserved gene, the Drosophila model system should prove useful for the determination of the ets gene function.     

Dipeptidyl peptidase III from rat liver cytosol: purification, molecular cloning and immunohistochemical localization

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of various rat tissues by immunohistochemical examination.