There is increasing molecular and antigenic evidence that Pneunocystis 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 13to 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.
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.
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. carinùi 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 107 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. caring 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. carinu 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 transferable between animals housed in the same room.
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.
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