Characterization and Cloning of <i>Pneumocystis carinii</i> Nucleic Acid

Worley, M; Ivey, Michael H.; Graves, Donald H.

doi:10.1111/j.1550-7408.1989.tb05809.x

Cited by 4 publications

(1 citation statement)

References 16 publications

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“…These compounds were selectively toxic to the parasite when compared to cultured mammalian cells, in contrast to the G.C selective minor groove binding chromomycin A 3 . The A.T selective minor groove binding compound pentamidine is also used in treatment of Pneumocystis carinii infection in AIDS patients [34], and these organisms also have an enriched A.T content of about 67% [35].…”

Section: Targeting Genome Contentmentioning

confidence: 99%

The Genome as a Drug Target Sequence Specific Minor Groove Binding Ligands

Turner¹,

Denny

2000

CDT

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The ability to target defined sequences on the DNA molecule would be of enormous benefit to the treatment of human disease. Towards this goal much research has been invested in examining the DNA binding and biological mechanisms of action of sequence selective minor groove binding ligands. These compounds act in a variety of ways to inhibit gene expression and DNA replication and also alter nuclear architecture. Concomitant with this, minor groove adducts formed by certain compounds are inefficiently removed by cellular DNA repair systems and are extremely cytotoxic. Additionally compounds targeting A.T rich DNA sequences have found clinical use in the treatment of particular parasitic infections.

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Section: Targeting Genome Contentmentioning

confidence: 99%

The Genome as a Drug Target Sequence Specific Minor Groove Binding Ligands

Turner¹,

Denny

2000

CDT

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Quantitation of Absolute Pneumocystis carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated from Infected Rat Lungs are Haploid Organisms

Wyder

Rasch

Kaneshiro

1998

J Eukaryotic Microbiology

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The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37 degrees C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G + C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carini carinii isolated from infected rat lungs are haploid organisms.

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Sequence Survey of the Genome of the Opportunistic Microsporidian Pathogen, Vittaforma corneae

Mittleider

Green

Mann

et al. 2002

J Eukaryotic Microbiology

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The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV-infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy-four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory-cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme previously had been identified only from prokaryotes. The 74 GSS included 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein-encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r = 0.79) and Shizosaccharomyces pombe (r = 0.70), but was markedly dissimilar to E. cuniculi (r = 0.19).

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Characterization and Cloning of Pneumocystis carinii Nucleic Acid

Cited by 4 publications

References 16 publications

The Genome as a Drug Target Sequence Specific Minor Groove Binding Ligands

The Genome as a Drug Target Sequence Specific Minor Groove Binding Ligands

Quantitation of Absolute Pneumocystis carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated from Infected Rat Lungs are Haploid Organisms

Sequence Survey of the Genome of the Opportunistic Microsporidian Pathogen, Vittaforma corneae

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