Jared M. Fischer

Evolution and Speciation of Pneumocystis

Studies during the last decade have revealed that the genus Pneumocystis contains many distinct organisms. Differences have been observed for their cell surfaces, the conditions required for their growth, and their genomes. The genetic and functional diversity exhibited by these organisms indicate Pneumocystis contains more than one species. Controversy surrounds this issue because Pneumocystis cannot be cultured and mating experiments cannot be performed. However, the degree of sequence variation seen between several gene orthologues suggested that DNA could be utilized to diagnose Pneumocystis species, which in turn, has indicated the need for revising its nomenclature. In 1976 Frenkel named the species in human P. jirovecii [6] under the zoological code and in 1999, he transferred it to the botanical code [7] thus validly applying two new species names, Pneumocystis jirovecii n. sp. and Pneumocystis carinii n. sp., for organisms derived from human and rat, respectively. The purpose of this article is to determine if a single phylogeny underlies the genealogies of several genes and to estimate the times of speciation.

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.

Evolutionary rate of ribosomal DNA in Pneumocystis species is normal despite the extraordinarily low copy-number of rDNA genes

THE three genes encoding the major RNA components found in ribosomes and the DNA regions that separate these three genes are commonly called ribosomal DNA, or rDNA. Three species of Pneumocystis, P. carinii, P. murina, and P. jirovecii, have been studied sufficiently to conclude that while their rDNA structure is normal, their rDNA copy number is decidedly unique. The vast majority of eukaryotes have dozens of copies, often hundreds, of rDNA. By contrast, studies using Southern blotting and PCR have suggested that there is a single set of rDNA genes in the two studied Pneumocystis species. The low copy number of rDNA in Pneumocystis species raised a question regarding the rate of evolution at this locus. To address this issue, we analyzed the 18S rDNA evolution in three Pneumocystis species and compared them to rates of 18S rDNA evolution in other eukaryotes. The data indicated that Pneumocystis 18S rDNA has evolved at a rate typical for eukaryotes, despite its low copy number. The evolutionary rate data also showed that the internal transcribed spacers (ITS) of P. jirovecii are not too unstable to be useful as epidemiological markers.

Visualizing loss of heterozygosity in living mouse cells and tissues.

Loss of heterozygosity (LOH) in somatic cells can contribute to the genesis of cancer, but little is known about the frequency with which LOH occurs in normal cells of the body. To detect LOH in situ, we studied mouse shYFP embryonic stem (ES) cells and cells of the intestinal epithelia derived from these ES cells. shYFP ES cells are heterozygous at the ROSA26 locus. One copy of the locus carries a gene encoding a yellow fluorescent protein (YFP), while the other copy harbors an shRNA gene that produces a short hairpin RNA (shRNA) molecule that causes degradation of YFP mRNA. Nearly all cells in shYFP populations were faintly fluorescent, but brightly fluorescent cells arose at a rate of approximately 10(-5)bright cells/generation. Bright cells lacked the gene encoding the shRNA and contained two copies of the YFP gene. Comparison of these results to previous data on LOH in ES cells that lacked interfering shRNA showed that LOH in shYFP cells was not influenced by the presence of the shRNA. Bright cells were also seen in intestinal villi of chimeric mice made by injecting blastocysts with shYFP cells. These data demonstrate that this approach can detect LOH and suggest that it will allow detection of LOH in a broad array of tissues and cell types in transgenic mice made from shYFP cells.

Mutation in aging mice occurs in diverse cell types that proliferate postmutation

To determine the relationship between aging, cell proliferation and mutation in different cell types, hearts, brains and kidneys from G11 PLAP mice between 1 week and 24 months of age were examined. Mutant cells were detected in tissue sections by staining for Placental Alkaline Phosphatase (PLAP) activity, an activity that marks cells that have sustained a frameshift mutation in a mononucleotide tract inserted into the coding region of the human gene encoding PLAP. The number of PLAP+ cells increased with age in all three tissues. The types of cells exhibiting a mutant phenotype included cells that are proliferative, such as kidney epithelial cells, and cells that do not frequently replicate, such as cardiac muscle cells and neurons. In the brain, PLAP+ cells appeared in various locations and occurred at similar frequencies in different regions. Within the cerebellum, PLAP+ Purkinje cell neurons appeared at a rate similar to that seen in the brain as a whole. PLAP+ cells were observed in kidney‐specific cell types such as those in glomeruli and collecting tubules, as well as in connective tissue and blood vessels. In the heart, PLAP+ cells appeared to be cardiac muscle cells. Regardless of tissue and cell type, PLAP+ cells occurred as singletons and in clusters, both of which increased in frequency with age. These data show that age‐associated accumulation of mutant cells occurs in diverse cell types and is due to both new mutation and proliferation of mutant cells, even in cell types that tend to not proliferate.