Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I.

A gene that specifies production of Escherichia coli DNA topoisomerase I (omega protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli omega protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX.     

Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli.

We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I. A family of plasmids expressing the yeast TOP1 gene or 5'-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E. coli topA mutant. These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling. Functional complementation of a conditional-lethal mutation in topA, which encodes E. coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA. We also show that approximately 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E. coli, however, is more sensitive to such deletions.     

Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide.

Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophilic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of approximately 630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.     

Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II.

Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli. One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome. Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message. Mutants in which 1.7 kb of the sequence is deleted are viable. Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant. These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II.     

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.     

Suppression of chromosome segregation defects of Escherichia coli muk mutants by mutations in topoisomerase I

Escherichia coli muk mutants are temperature-sensitive and produce anucleate cells. A spontaneously occurring mutation was found in a DeltamukBkan mutant strain that suppressed the temperature-sensitive phenotype and mapped in or near topA, the gene that encodes topoisomerase I. Previously characterized topA mutations, topA10 and topA66, were found to be general suppressors of muk mutants: they suppressed temperature sensitivity and anucleate cell production of cells containing null or point mutations in mukB and null mutations in mukE or mukF. The suppression correlated with excess negative supercoiling by DNA gyrase, and the gyrase inhibitor, coumermycin, reversed it. Defects in topA allow 99% of cell division events in muk null mutants to proceed without chromosome loss or loss of cell viability. This observation imposes important limitations on models for Muk activity and is consistent with a role for MukBEF in chromosome folding and DNA condensation.     

Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3).

A cloned segment of yeast DNA containing the structural gene for imidazoleglycerolphosphate dehydratase (D-erythro-imidazoleglycerolphosphate hydro-lase, EC 4.2.1.19) is transcribed and translated in Escherichia coli with sufficient fidelity to produce functional enzyme. This segment of yeast DNA was isolated as a viable molecular hybrid of bacteriophage lambda (lambdagt-Sc2601) which complements a nonrevertible hisB auxotroph of E. coli lacking dehydratase activity. The equivalent segments of DNA cloned from two independent his3 mutants of yeast lacking IGP dehydratase activity do not complement the hisB auxotroph. The two nonfunctional his3 alleles cloned in bacteriophage lambda can be recombined in E. coli to generate a hybrid phage which complements the hisB auxotroph. The dehydratase activity produced in E. coli by the cloned segment of yeast DNA strongly resembles the activity found in yeast.     

Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli

Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.     

Alternate pathways of DNA replication: DNA polymerase I-dependent replication.

We have previously shown that some Escherichia coli [derivatives of strain HS432 (polA1, polB100, polC1026)] can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity. We demonstrate here that this strain of E. coli becomes temperature-resistant upon the introduction of a normal gene for DNA polymerase I or suppression of the polA1 nonsense mutation. Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene. Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E. coli. HS432 contains a transducible locus (which we term pcbA) that can support an alternate pathway in other E. coli strains, so the effect of suppression of polCts is a general one.     

Adenovirus infection elevates levels of cellular topoisomerase I.

We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme.     

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.     

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.     

Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria.

Construction of physical maps of genomes by pulsed-field gel electrophoresis requires enzymes which cut the genome into an analyzable number of fragments; most produce too many fragments. The enzyme I-Ceu I, encoded by a mobile intron in the chloroplast 23S ribosomal RNA (rrl) gene of Chlamydomonas eugametos, cuts a 26-bp site in the rrl gene. This enzyme digests DNA of Salmonella typhimurium at seven sites, each corresponding to one of the rrl genes of the rrn operons, but at no other site. These seven fragments were located on the previously determined Xba I physical map, and the I-Ceu I sites, and thus the rrn genes of S. typhimurium, were mapped on the 4800-kb chromosome. Escherichia coli K-12 also yields seven fragments of sizes similar to those of S. typhimurium, indicating conservation of rrn genes and their location, and a chromosome size of 4600 kb. The sizes of the E. coli fragments are close to the size predicted from restriction maps and nucleotide sequence. The I-Ceu I maps of Salmonella enteritidis, Salmonella paratyphi A, B, C, and Salmonella typhi were deduced after digesting genomic DNA and I-Ceu I and probing with DNA of S. typhimurium; the data indicated strong conservation of rrn gene number and position and genome sizes up to 4950 kb. Digestion of DNA of other bacteria (species of Haemophilus, Neisseria, Proteus, and Pasteurella) suggested that only rrn genes are cut in all these species. I-Ceu I digestion followed by pulsed-field gel electrophoresis is a powerful tool for determining genome structure and evolution.     

Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II.

We have isolated the gene coding for the Drosophila type II DNA topoisomerase by immunochemically screening a Drosophila cDNA library constructed with a phage lambda expression vector, lambda gt11. The identity of the cloned gene is confirmed by the analysis of an antigenic fusion protein produced in Escherichia coli and by the in vitro translation of its RNA. The gene is 5.1 kilobases in length, the expected size for a gene encoding topoisomerase II (Mr 170,000), and it is divided into five exons. By in situ hybridization to the polytene chromosomes from salivary glands, we have mapped it to chromosome 2L at 37D.     

编码弓形虫表面抗原P30基因的克隆及在E.coli中的表达

目的　构建编码弓形虫RH株表面抗原P30基因重组表达质粒 ,初步观察P30基因在E coli表达。方法　将P30基因定向克隆到分支杆菌 -大肠杆菌穿梭表达质粒热休克蛋白 70 (hsp70 )起动基因的下游的多克隆位点 ,构建重组表达质粒pBCG -P30 ;采用亚克隆技术 ,将含P30和hsp70起动基因的复合片段 ,插入表达载体 pBK -CMV质粒 ,转化大肠杆菌DH5α ,在卡那霉素阳性LB培养基平板筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒 pBK -P30转化大肠杆菌 ,IPTG诱导表达后进行SDS -PAGE和Westernboltting分析。 结果　 1)阳性重组质粒 pBCG -P30、pBK -P30经酶切和PCR鉴定 ,与预期的结果相符合。 2 )序列测定证实克隆的基因为编码P30抗原的基因。 3)P30基因在大肠杆菌诱导表达后获得4 5kDa融合蛋白 ,此抗原未被弓形虫高免兔血清识别。结论　成功构建编码弓形虫表面抗原P30重组表达质粒 ,并在大肠杆菌中获得表达 ,为弓形虫DNA疫苗的研制奠定基础     

Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.

Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.