Saundra L. Stringer

Pneumocystis carinii: sequence from ribosomal RNA implies a close relationship with fungi

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.

Molecular Genetic Distinction of Pneumocystis carinii from Rats and Humans

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 α‐tubulin genes from the two P. carinii were strikingly different and the base composition of the α‐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.

Genes Encoding Antigenic Surface Glycoproteins in Pneumocystis from Humans

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.

A Tandem Repeat of Rat‐derived Pneumocystis carinii Genes Encoding the Major Surface Glycoprotein

ABSTRACT. 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.

A tandem pair of Leishmania donovani cation transporting ATPase genes encode isoforms that are differentially expressed.

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.

Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

BackgroundLoss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cells phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors.ResultsAs a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6.ConclusionAllelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.