Assuntos
Genes Fúngicos , Pneumocystis/genética , RNA Fúngico/genética , RNA Ribossômico/genética , Animais , DNA Fúngico/genética , DNA Ribossômico/genética , Variação Genética , Família Multigênica , Pneumocystis/classificação , Pneumocystis/isolamento & purificação , Ratos , Mapeamento por Restrição , Especificidade da EspécieAssuntos
Genes Fúngicos , Pneumocystis/genética , Animais , Furões , Humanos , Camundongos , Filogenia , Pneumocystis/classificação , Pneumocystis/isolamento & purificação , Pneumonia por Pneumocystis/microbiologia , Reação em Cadeia da Polimerase , RNA/genética , RNA Fúngico/genética , RNA Mitocondrial , RNA Ribossômico 18S/genética , Ratos , Especificidade da Espécie , Suínos , Timidilato Sintase/genéticaRESUMO
A fragment from the genome of rat-derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non-identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5' and 3' untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5' end of MSG and downstream of the 3' end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos/genética , Glicoproteínas de Membrana/genética , Pneumocystis/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Fúngico/análise , Terapia de Imunossupressão , Pulmão/microbiologia , Dados de Sequência Molecular , Ratos , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.
Assuntos
Antígenos de Fungos/genética , Genes Fúngicos , Glicoproteínas de Membrana/genética , Pneumocystis/genética , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Fúngico/análise , Humanos , Pulmão/microbiologia , Dados de Sequência Molecular , Pneumocystis/imunologia , Pneumocystis/isolamento & purificação , Infecções por Pneumocystis/etiologia , Infecções por Pneumocystis/microbiologia , Ratos , Ratos Endogâmicos BN , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the alpha-tubulin genes from the two P. carinii were strikingly different and the base composition of the alpha-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.
Assuntos
Pneumocystis/classificação , Pneumocystis/genética , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Primers do DNA , DNA Fúngico/química , DNA Fúngico/isolamento & purificação , Variação Genética , Humanos , Íntrons , Pulmão/microbiologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Pneumocystis/isolamento & purificação , Infecções por Pneumocystis/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
There is increasing molecular and antigenic evidence that Pneumocystis carinii organisms isolated from humans, ferrets, and rats are different species. In contrast, little is known about the extent of genetic diversity among P. carinii strains found within a single mammalian species. In the present study, electrophoretic karyotypes were obtained from P. carinii prepared from 10 chronically immunosuppressed rat colonies to investigate diversity at the chromosomal level. Most organism preparations produced patterns with 13 to 15 bands, but as many as 24 bands were observed in a few preparations. All bands separated between 700 and 300 kbp. Four distinct karyotype forms emerged from among the 13- to 15-band karyotypes of the 10 colonies sampled. Form 1 was shared by five rat strains from two vendors; form 2 was shared by two rat strains from the same vendor; and forms 3 and 4 were unique to their vendor colonies. Within a given rat colony, most rats harbored the same P. carinii karyotype. A survey of selected rat colonies showed that the karyotype within a vendor colony could remain stable over a period of 2 to 3 years. Hybridization of the blotted karyotypes with a repetitive DNA element isolated from rat-derived P. carinii and with single-copy gene probes showed that every chromosome in the karyotypes contained some repetitive DNA, and there was a general size concordance among the chromosomes carrying the unique gene loci. Differences in gene sequences, electrophoretic karyotypes, and hybridization profiles suggested that the immunosuppressed rats were infected by genetically distinct P. carinii strains. A provisional system of nomenclature for P. carinii that will permit differentiation of P. carinii organisms from the same mammalian host is discussed. These data show that all rats were not infected by a single type of P. carinii, that pulsed-field gradient electrophoresis can detect sufficient genetic diversity among the organism preparations to allow for characterization of the organisms, and that the genome of the organism within the rat host is relatively stable over time.
Assuntos
Variação Genética , Infecções por Pneumocystis/veterinária , Pneumocystis/genética , Ratos/microbiologia , Doenças dos Roedores/microbiologia , Animais , Eletroforese , Cariotipagem , Masculino , Hibridização de Ácido Nucleico , Infecções por Pneumocystis/microbiologiaRESUMO
Pneumocystis carinii pneumonia is an oftentimes fatal infection for hosts in an immunocompromised state. The disease occurs in a wide variety of mammals, but the etiologic agent of this disease has been referred to as P. carinii regardless of the host species. However, even within a single host species, such as laboratory rats, distinct varieties of P. carinii have been identified from differences in the electrophoretic migration of chromosomes in agarose gels. Here we present evidence indicating that some laboratory rats can contain two different genetic variants of P. carinii that differ not only in electrophoretic karyotype but also in the presence of a particular repeated DNA sequence, in the presence of an intron in the 18S ribosomal RNA gene, and in the sequence of part of the 18S rRNA gene. Most of the rat colonies studied were infected with P. carinii that contained the repeated DNA and the 18S rRNA gene intron. The other type of rat-derived P. carinii, which lacked the repeated DNA and the intron in the 18S rRNA gene, was found as a coinfection with the first. Parasite populations from different coinfected rats contained the two variants in different proportions.
Assuntos
Pneumocystis/genética , Pneumocystis/isolamento & purificação , Ratos/microbiologia , Animais , Sequência de Bases , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Variação Genética , Cariotipagem , Masculino , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Sequências Repetitivas de Ácido NucleicoRESUMO
Chromosome-specific DNA probes were isolated to identify a particular chromosome in various karyotype band patterns of Pneumocystis. Bands in two P. carinii karyotype patterns hybridized to various DNA probes indicated that homologous chromosomes are of relatively similar sizes in the two P. carinii strains.
Assuntos
Genoma Fúngico , Pneumocystis/genética , Sondas de DNA , Variação Genética , CariotipagemRESUMO
A 16-kb DNA fragment designated Rp3-1 and cloned from the genome of rat-derived Pneumocystis carinii was found to contain sequences that were repeated approximately 70 times per genome. The repeated sequences in Rp3-1 spanned at least 10.4 kb. Sequences in Rp3-1 were present on each of the 16 P. carinii chromosomes resolved by field inversion gel electrophoresis. Most of the P. carinii genomic sequences homologous to those in the Rp3-1 clone appeared to be as long as those in the Rp3-1 clone but were highly polymorphic with respect to restriction enzyme cleavage sites. The Rp3-1 DNA fragment appears to be a member of a family of large, degenerate, dispersed repeats.
Assuntos
DNA Fúngico/genética , Pneumocystis/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Clonagem Molecular , DNA Fúngico/isolamento & purificação , Ligação Genética , Pneumonia por Pneumocystis/microbiologia , Polimorfismo de Fragmento de Restrição , Ratos , Ratos Endogâmicos , Mapeamento por RestriçãoRESUMO
Pulsed-field gel electrophoresis techniques were used to examine the chromosomes of Pneumocystis carinii isolated from laboratory rats and two human subjects. P. carinii organisms isolated from each of four rat colonies and from two patients each produced a distinct band pattern, but in all cases the bands ranged in size from 300 to 700 kilobase pairs. P. carinii from three rat colonies produced patterns containing 15 prominent bands. Of these 15 bands, 2 stained more intensely than would be expected of bands of their size, suggesting that the P. carinii haploid genome contains 17 to 19 chromosomes. Summing the molecular sizes of the bands and accounting for staining intensities suggested that the haploid genome of rat-derived P. carinii contains on the order of 10(7) base pairs. Human-derived P. carinii produced patterns containing 10 to 12 bands which appeared to be similar to the 15-band patterns seen in rat-derived P. carinii with respect to the size range of the bands. P. carinii from the fourth rat colony produced a more complex band pattern containing approximately 22 bands, most of which appeared to comigrate with the bands present in one of the 15-band P. carinii patterns, suggesting that these animals were simultaneously infected by two different varieties of P. carinii. Hybridization experiments using oligonucleotide probes specific for the P. carinii 18S rRNA gene supported this possibility. The band pattern of P. carinii derived from a given rat colony was generally stable over time. P. carinii band patterns were not strictly rat strain specific and appeared to be transferrable between animals housed in the same room.
Assuntos
Cromossomos Fúngicos/ultraestrutura , Pneumocystis/genética , Animais , DNA Fúngico/análise , Eletroforese , Variação Genética , Humanos , Cariotipagem , Masculino , Peso Molecular , Pneumonia por Pneumocystis/epidemiologia , Pneumonia por Pneumocystis/genética , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/análise , Ratos , Ratos Endogâmicos , Sequências Repetitivas de Ácido Nucleico , Estados Unidos/epidemiologiaRESUMO
Pneumocystis carinii is the etiologic agent of a lethal pneumonia which occurs in patients with the acquired immune deficiency syndrome (AIDS) and other immunocompromised hosts. The basic biochemical and genetic characteristics of P. carinii are poorly understood and its taxonomic classification as a protozoan is uncertain. To address the taxonomic question, a method was developed for the extraction of total RNA from P. carinii. Denaturing agarose gel electrophoresis showed the two ribosomal RNA species of P. carinii to be similar in size to those of other lower eukaryotes, including Saccharomyces cerevisiae. Three portions of the small ribosomal RNA of P. carinii were sequenced by reverse transcription from oligonucleotide primers. Three hundred seventy-two nucleotides of sequence were obtained. The sequence derived from P. carinii RNA contained regions that previous phylogenetic studies have shown to be highly variable among species, as well as regions that are highly conserved. Comparison of the P. carinii sequence to corresponding sequences of organisms from other taxa showed the P. carinii sequence to be more similar to sequences from the fungi (Saccharomyces cerevisiae, Neurospora crassa, Candida albicans, and Cryptococcus diffuens) than to protozoan sequences. These data suggest that P. carinii is more closely related to fungi than to protozoa.
Assuntos
Fungos/classificação , Pneumocystis/classificação , RNA Ribossômico/genética , Acanthamoeba/classificação , Acanthamoeba/genética , Animais , Sequência de Bases , Candida albicans/classificação , Candida albicans/genética , Cryptococcus/classificação , Cryptococcus/genética , Eletroforese em Gel de Ágar , Fungos/genética , Dados de Sequência Molecular , Neurospora crassa/classificação , Neurospora crassa/genética , Filogenia , Plasmodium berghei/classificação , Plasmodium berghei/genética , Pneumocystis/genética , RNA/análise , RNA/isolamento & purificação , RNA Fúngico/genética , RNA Ribossômico/isolamento & purificação , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Toxoplasma/classificação , Toxoplasma/genética , Transcrição GênicaRESUMO
The second gene (ATPase 1b) of a tandem pair of cation transporting ATPases from Leishmania donovani was cloned and sequenced. The sequence of this gene was very similar to its upstream neighbor (ATPase 1a). Both genes contained a 2922 base open reading frame capable of encoding a protein of 974 amino acids. The genes differed at 34 nucleotide base positions, predicting 20 amino acid differences between the two peptides. These changes were clustered at the carboxy terminus with 15 changes occurring in the COOH-terminal 37 amino acids. However, these changes did not alter the highly charged nature of the carboxy terminus observed in ATPase 1a. The sequence was also conserved for 73 bases upstream of ATPase 1a and 1b but downstream conservation was limited to 15 bases beyond the termination codon. RNA from ATPase 1a was 5.2 kb and was present in both developmental forms of Leishmania. By contrast the ATPase 1b gene expressed a 5.75 kb transcript which was much more abundant in the amastigote form of Leishmania than in the promastigote form.
