Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans

Restrepo, Blanca I.; Barbour, Alan G.

doi:10.1128/jb.171.10.5596-5600.1989

Cited by 37 publications

(16 citation statements)

References 27 publications

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“…Strains were grown in YPG (yeast extract 1%, bactopeptone 2%, glucose 2%) with added penicillin G (20 U ml x1 ) and streptomycin sulphate (40 U ml x1 ) at 30 uC with shaking for 24 h. The isolation of Trichosporon genomic DNA was based on the method of Restrepo and Barbour [12] used in Cryptococcus neoformans DNA extraction with some slight modi®cations [13]. In brief, cells were grown in potato glucose broth (PDB) instead of Brain heart infusion glucose (BHG) medium and the lysing solution contained 0.05 M M EDTA instead of 0.5 M M EDTA.…”

Section: Dna Isolationmentioning

confidence: 99%

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope

Passo

Pernice

Celeste

et al. 2001

Mycoses

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Two cases of oesophageal trichosporonosis due to a suspected nosocomial infection are reported. Both the patients were immunocompetent and had undergone an endoscopic examination on the same day. Six strains of Trichosporon were isolated: three strains from the oesophageal biopsy of the first patient, one strain from the endoscopic forceps, one from the air in the endoscopy room, and one from the oesophageal biopsy of the second patient. The nosocomial nature of the infection and the role of the endoscopic forceps in transporting the micro-organism was suspected, but the morphology and physiology of the isolated strains did not confirm such hypothesis. To elucidate the nature of the infection and the genetic similarities of the strains isolated, all strains were typed with RFLPs of the rDNA fragment and with RAPD. The results of RAPD using primer (GTG)5 (GACA)4, M13 core sequence, and the 15-mer oligonucleotide GAGGGTGGXGGXTCT indicated the molecular identity of three strains supporting the hypothesis concerning a transport of the aetiological agent from the first patient to the second and that the carrier was the forceps of the endoscopic device.

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Section: Dna Isolationmentioning

confidence: 99%

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope

Passo

Pernice

Celeste

et al. 2001

Mycoses

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“…by analysis of enzymatically amplified rDNA fragments. The various rRNAs and their respective DNA coding regions are known for their value as evolutionary markers, since they contain regions of both high and low sequence variability (5 RNAs along with their respective spacer segments (18). In this study, we demonstrate the utility of restriction analysis using PCR products for (i) amplification and restriction mapping of rDNA from Cryptococcus species, (ii) identification of species-and strain-specific RFLPs, (iii) linking of anamorphic and teleomorphic life stages, and (iv) phylogenetic analysis.…”

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confidence: 99%

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species

1990

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Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel ap h t uses the polymerase chain reaction (PCR) Several limiting features of conventional restriction analyses using Southern blot hybridization for routine identification and systematics are a requirement for relatively clean DNA samples as well as substantial time and resources necessary for preparing DNA hybridization membranes and probes. Sensitivity of Southern blots is also sometimes affected by poor signal/noise ratios caused by poor hybridization of heterologous probes or high background signals on blotting membranes. To circumvent some of these potential problems, we have used an approach that allows direct analysis of a desired target sequence without the necessity of blotting or hybridization. Our approach utilizes the polymerase chain reaction (PCR) to enzymatically amplify target sequences in vitro by a factor of over 108, starting from minute amounts of genomic DNA template (19,21). The level of sensitivity, specificity, and adaptability of the PCR has resulted in many applications, including efficient cloning, rapid direct sequencing, and highly sensitive detection (2,12,19,22). The basic PCR protocol involves the use of two oligonucleotide primers (which flank a target DNA sequence) to initiate DNA synthesis on opposing strands of a target sequence, using a heat-stable DNA polymerase. Repeated cycles (20 to 30) of denaturation, primer annealing, and DNA synthesis result in a theoretical doubling of target sequence during each cycle. Automation of the PCR by using a thermal cycler device allows simultaneous amplification of DNA from many samples, which can then be used directly for analysis.In this study, we examined DNA sequence variation in the rDNA of several Cryptococcus spp. by analysis of enzymatically amplified rDNA fragments. The various rRNAs and their respective DNA coding regions are known for their value as evolutionary markers, since they contain regions of both high and low sequence variability (5

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“…A conserved ribosomal gene probe provided by K.-J. Kwon-Chung (National Institutes of Health, Bethesda, MD, USA; Restrepo & Barbour, 1989), and the conserved 600 bp PCR-amplified fragment of the CHS gene of C. immitis derived from this study were used as DNA probes. The hybridization protocol was performed as previously described (Pan & Cole, 1992).…”

Section: Dna Extractionmentioning

confidence: 99%

Evidence for a phylogenetic connection between Coccidioides immitis and Uncinocarpus reesii (Onygenaceae)

Pan

Sigler²,

Cole³

1994

Microbiology

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Coccidioides immitis is an anomaly amongst the human systemic fungal pathogens. Its unique parasitic cycle has contributed to confusion over its taxonomy. Early investigators mistakenly suggested that the pathogen is a protist, while others agreed it to be a fungus but placed it in four different divisions of the Eumycota. The taxonomy of C. immitis is still unresolved. Ultrastructural examinations of its parasitic and saprobic phases have revealed features that are diagnostic of the ascomycetous fungi. Moreover, striking similarities between the kind of asexual reproduction (i.e. arthroconidium formation) of this pathogen and certain anamorphic and teleomorphic members of the genus Malbranchea have suggested a close relationship. Teleomorphs of these Malbranchea species are members of the Onygenaceae (Order, Onygenales). This family also includes teleomorphs of two human respiratory pathogens, Histoplasma capsulatum and Blastomyces dermatitidis. Although the 185 rRNA gene sequences (1713 bp) of these two pathogenic forms differ from that of C. immitis by only 35 and 33 substitutions, respectively, their mode of conidiogenesis is characterized by production of solitary aleurioconidia rather than alternate arthroconidia. In this study we have used characters derived from biochemical, immunological and molecular analyses to compare relatedness between C. immitis, H. capsulatum, B. dermatitidis, and six non-pathogenic species of Malbranchea (the Malbranchea states of Uncinocarpus reesii and Auxatthron zuffianum, as well as M. albolutea, M. dendritica, M. filamentosa and M. gypsea). Evidence is presented which supports inclusion of C. immitis in the Onygenaceae, and indicates that a close phylogenetic relationship exists between the Malbranchea state of U. reesii and this respiratory pathogen.

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Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans

Cited by 37 publications

References 27 publications

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope

Transmission of Trichosporon asahii oesophagitis by a contaminated endoscope

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species

Evidence for a phylogenetic connection between Coccidioides immitis and Uncinocarpus reesii (Onygenaceae)

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