Molecular cloning and expression of the <i>Candida albicans TOP2</i> gene allows study of fungal DNA topoisomerase II inhibitors in yeast

Keller, Beatrice A.; Patel, Sandhiya; Fisher, L. Mark

doi:10.1042/bj3240329

Cited by 16 publications

(9 citation statements)

References 44 publications

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“…The DNA topoisomerase II gene is present in all eukaryotes and its sequence is well conserved and composed of highly homologous and species-specific regions (15,16). The characteristics of this gene are an advantage to a target gene not only in the study of phylogenetic relationships among fungal species and evolutionary modes of fungal genes (14), but also in molecular biology-based identification of a broad range of fungal species (13).…”

Section: Resultsmentioning

confidence: 99%

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Kanbe

Yamaki

Kikuchi

2002

Microbiology and Immunology

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For PCR‐based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species‐specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species‐specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species‐specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.

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Section: Resultsmentioning

confidence: 99%

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Kanbe

Yamaki

Kikuchi

2002

Microbiology and Immunology

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“…Of these, alanine-substitution at Arg-475 and Lys-477, both of which are present in a highly conserved motif PLRGKILN (positions underlined), had previously been done in connection with a search of mutations that might lead to resistance to the antitumor agent amsacrine (15); alanine substitution at neither one of the adjacent pair of basic residues had a major effect on the catalytic activities of the enzyme. Asp-451, which lies within a highly conserved motif EGDSA (position underlined), was not selected because of the presence of a leucine at this position in Candida albican DNA topoisomerase II (16). Alanine substitution was carried out at each of the remaining nine positions.…”

Section: Resultsmentioning

confidence: 99%

Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases

Liu

Wang

1999

Proc. Natl. Acad. Sci. U.S.A.

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Studies of yeast DNA topoisomerase II with various alanine-substitution mutations provide strong biochemical support of a recent hypothesis that the type IA and IIA DNA topoisomerases act similarly in their cleavage and rejoining of DNA. DNA breakage and rejoining by either a type IA or a type IIA enzyme are shown to involve cooperation between a DNA-binding domain containing the active-site tyrosine and a Rossmann fold containing several highly conserved acidic residues. For a homodimeric type IIA enzyme, cooperation occurs in trans: the active-site tyrosine in the DNA-binding domain of one protomer cooperates with several residues in the Rossmann fold as well as other regions of the other protomer.The DNA topoisomerases are enzymes that introduce transient breaks in DNA strands to permit their interpenetration (for a review, see ref. 1). These enzymes are divided into two types: the type I enzymes transiently break one DNA strand at a time for the passage of a second strand or perhaps a duplex DNA, and the type II enzymes transiently break both strands of one DNA double helix for the passage of a second DNA double helix. Extensive studies indicate that the type I enzymes can be further divided into two subfamilies, IA and IB, which are distinct in amino acid sequences and reaction mechanisms. The amino acid sequences of a large number of phylogenetically diverse type II DNA topoisomerases suggest that they can also be grouped into two subfamilies, IIA and IIB: the former includes eukaryotic DNA topoisomerase II, bacterial gyrase, and phage T4 DNA topoisomerase, and the latter, archaeal DNA topoisomerase VI (2).In recent years, the three-dimensional structures of several large fragments of the type IA, IB, and IIA subfamilies of DNA topoisomerases have been determined. As expected from the amino acid sequences and mechanistic properties of these enzymes, the overall tertiary structures of enzymes of different subfamilies appear to be very different (reviewed in ref.3). Recent comparisons of the type IA and IIA enzymes reveal, however, that there are common structural elements in both groups (4, 5). In the crystal structure of a 67-kDa fragment of a type IA enzyme, Escherichia coli DNA topoisomerase I (6), there are two domains that are similar to the DNA-binding domain of the E. coli catabolite activator protein (CAP). These CAP-like domains, one in domain III and the other in domain IV of the 67-kDa fragment, are related by a pseudo dyad axis (7). A CAP-like fold is also seen in the crystal structures of a 92-kDa fragment of yeast DNA topoisomerase II (8) and a 59-kDa fragment of the GyrA subunit of E. coli gyrase (9

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“…carrying highly homologous genes, so that the number of species that can be identified to the species level is limited. In contrast to these genes, the DNA topoisomerase II gene is present in all eukaryotes and its sequence is well conserved and composed of highly homologous regions and species-specific regions (Keller et al, 1997). The characteristics of this gene are of enormous advantage to a target gene in molecular biology-based identification for a broad range of fungal species.…”

Section: Discussionmentioning

confidence: 99%

“…The DNA topoisomerase II gene is present in all eukaryotes, and its nucleotide sequence is composed of highly conserved regions separated by species-specific regions (Keller et al, 1997). The sequence analysis of the DNA topoisomerase II (DNA gyrase) gene in bacterial species was applied not only to understand phylogenetic relationships but also for development of a diagnostic identification system of broad species of medically important bacteria by PCR (Huang, 1996).…”

Section: Introductionmentioning

confidence: 99%

PCR‐based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes

Kanbe

Horii

Arishima

et al. 2002

Yeast

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For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCRbased identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.

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Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

Cited by 16 publications

References 44 publications

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases

PCR‐based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes

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