Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.     

卫氏并殖吸虫基因片段的克隆与鉴定

目的从卫氏并殖吸虫成虫cDDA文库中筛选并鉴定可用于免疫诊断和免疫预防的基因克隆。方法采用预吸收成虫抗原免疫兔血清(IRS,多抗)筛选阳性克隆,经PCR扩增后测定其插入片段的大小。序列分析后,对筛选的重组子进行双酶切,亚克隆入表达载体pRESETB,并转化大肠杆菌BL-21细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,对表达产物进行SDS-PAGE和Westernblotting分析鉴定。结果所筛选的阳性克隆插入片段约800bp。DNA序列分析显示其为编码半胱氨酸蛋白酶族组织蛋白酶L的基因序列。Pw-2重组子特异性表达产物约为32kDa,可被卫氏并殖吸虫免疫兔血清特异地识别。结论从卫氏并殖吸虫成虫cDNA文库筛选的重组质粒Pw-2编码属于半胱氨酸蛋白酶族组织蛋白酶L。     

The 37/40-kilodalton autoantigen in insulin-dependent diabetes mellitus is the putative tyrosine phosphatase IA-2.

Major targets for autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) include tryptic fragments with a molecular mass of 37 kDa and/or 40 kDa of a pancreatic islet cell antigen of unknown identity. The assay identifying autoantibodies against the 37/40-kDa antigen in human sera is based on the immunoprecipitation of 35S-labeled rat insulinoma cell proteins with sera from IDDM patients, followed by limited trypsin digestion of the immunoprecipitated material. To identify cDNA clones coding for the 37/40-kDa antigen, we have screened a cDNA expression library from rat insulinoma cells with a serum from an IDDM patient that precipitated the 37/40-kDa antigen in our assay. Among the cDNA products that reacted with the IDDM serum, we identified one cDNA clone whose open reading frame encodes a protein with a predicted mass of 105 kDa that we termed "ICA105" for 105-kDa islet cell antibody. The deduced amino acid sequence has high homology to a recently cloned putative tyrosine phosphatase IA-2 from human and mouse cDNA libraries. Translation of the cDNA in vitro results in a polypeptide with the expected molecular mass of 105 kDa. The evidence that ICA105 is indeed the precursor of the 37/40-kDa tryptic fragments is based on the following three results: (i) Sera from IDDM patients containing autoantibodies to the 37/40-kDa antigen precipitate the in vitro translated polypeptide, whereas sera from healthy subjects as well as sera from IDDM patients not reactive with the 37/40-kDa antigen do not precipitate the cDNA product. (ii) Immunoprecipitation of the in vitro translated protein with sera containing autoantibodies to the 37/40-kDa antigen followed by limited trypsin digestion of the precipitated proteins results in a 40-kDa polypeptide. (iii) The protein derived from our cDNA but not from an unrelated control cDNA clone can block immunoprecipitation of the 37/40-kDa antigen from a labeled rat insulinoma cell extract. The availability of the cloned 37/40-kDa antigen should facilitate the identification of individuals at risk of IDDM with increased accuracy. Furthermore, the identification of the 37/40-kDa antigen as the putative tyrosine phosphatase IA-2 is of relevance in elucidating the role of this antigen in the development of IDDM.     

Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae.

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.     

Identification of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity associated with the mouse plasma cell differentiation antigen PC-1.

The protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was purified from MOPC 315 plasmacytoma cells. A single SDS/PAGE-purified 115-kDa protein band was used to produce a rabbit polyclonal antiserum. This antibody preparation precipitated alkaline phosphodiesterase I activity, indicating that the SDS/PAGE-purified protein was nucleotide pyrophosphatase/alkaline phosphodiesterase I. When used for Western blot analysis, the antiserum detected a 115-kDa protein as well as a 220-kDa protein band. Multiple overlapping cDNA clones were isolated from a cDNA expression library screened with this anti-nucleotide pyrophosphatase/alkaline phosphodiesterase I antiserum. Sequence analysis indicated that the isolated cDNA clones encoded PC-1, a murine plasma cell differentiation antigen. To confirm the suspected enzymatic identity of PC-1, a recombinant PC-1 fusion protein was expressed in bacteria, purified, and used to produce another rabbit polyclonal antiserum. This antiserum likewise immunoprecipitated alkaline phosphodiesterase I activity and recognized the 115-kDa and 220-kDa proteins in Western blot analyses of cell extracts. Furthermore, expression of nucleotide pyrophosphatase/alkaline phosphodiesterase I corresponded directly with mRNA and protein levels of PC-1 in cells known to express different levels of nucleotide pyrophosphatase/alkaline phosphodiesterase I activity. Finally, steroid induction of enzymatic activity was mirrored by levels of PC-1 mRNA and protein expression. Together, these data indicate that the plasma cell differentiation antigen PC-1 is a membrane-bound enzyme, nucleotide pyrophosphatase/alkaline phosphodiesterase I.     

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.     

Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase.

We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in lambda Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxyl-terminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.     

Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 20q12-13.2.

cDNA clones of the human TOP1 gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage lambda libraries expressing human cDNA segments, using rabbit antibodies raised against purified HeLa DNA topoisomerase I. Hybridization patterns between the cloned cDNA sequences and human cellular DNA and cytoplasmic mRNAs indicate that human TOP1 is a single-copy gene. The chromosomal location of the gene has been mapped to the long arm of chromosome 20, in the region q12-13.2, by hybridization of a radioactively labeled TOP1 cDNA probe to human metaphase chromosomes and to a panel of rodent-human somatic hybrids retaining overlapping subsets of human chromosomes.     

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.