James R. Stringer

Embryonic stem cells and somatic cells differ in mutation frequency and type.

Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss/reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.

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.

Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas

The RET/PTC3 rearrangement is formed by fusion of the ELE1 and RET genes, and is highly prevalent in radiation-induced post-Chernobyl papillary thyroid carcinomas. We characterized the breakpoints in the ELE1 and RET genes in 12 post-Chernobyl pediatric papillary carcinomas with known RET/PTC3 rearrangement. We found that the breakpoints within each intron were distributed in a relatively random fashion, except for clustering in the Alu regions of ELE1. None of the breakpoints occurred at the same base or within a similar sequence. There was also no evidence of preferential cleavage in AT-rich regions or other target DNA sites implicated in illegitimate recombination in mammalian cells. Modification of sequences at the cleavage sites was minimal, typically involving a 1 – 3 nucleotide deletion and/or duplication. Surprisingly, the alignment of ELE1 and RET introns in opposite orientation revealed that in each tumor the position of the break in one gene corresponded to the position of the break in the other gene. This tendency suggests that the two genes may lie next to each other but point in opposite directions in the nucleus. Such a structure would facilitate formation of RET/PTC3 rearrangements because a single radiation track could produce concerted breaks in both genes, leading to inversion due to reciprocal exchange via end-joining.

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.

Genetics of Surface Antigen Expression in Pneumocystis carinii

ABSTRACT This article reviews the molecular genetic data pertaining to the major surface glycoprotein (MSG) gene family of Pneumocystis carinii and its role in surface variation and compares this fungal system to antigenic variation systems in the protozoanTrypanosoma brucei and the bacteriaBorrelia spp.

New Nomenclature for the Genus Pneumocystis

Approximately 50 individuals attended the round-table discussion session on Pneumocystis nomenclature. The group discussed the desirability and appropriateness of retaining the currently used tripartite nomenclature system, as opposed to assigning new species names to organisms that seem to be too different to be accommodated within a single species. The group unanimously endorsed a proposal to rename organisms currently known as special forms of P. carinii as species in the genus fnewwcystis. Whereas the discussion at the round-table session led to a clear endorsement of the proposal to apply new species names, it should be noted that not all of the ideas and opinions expressed in this article were discussed during the meeting. Furthermore. time and distance prohibited providing every interested party the opportunities to either contribute to this text, or to critique it prior to publication. Therefore, the content of this article should not be viewed as having been explicitly endorsed by everyone at the meeting, or other interested individuals in the community of Pnewnocystis researchers. With this disclaimer in mind, the purposes of this article are confined to the following: 1) explain the rationale for changing the nomenclature, 2) provide guidelines for recognizing new species and assigning new species names. These guidelines may become conventions in the future. but it is more probable that modifications will be needed as the complexity of the genus becomes more completely described. Indeed, recent new data have already clouded the issue to some extent. Analysis of one genetic locus in Pnewnocystis organisms from a large number of primates (i.e., from a single taxonomic Order) showed that sequence divergence can be much less than it is when Pneumocystis organisms from hosts in different Orders of mammals are compared. Thus. the consensus in favor of recognizing new species emerged contemporaneously with new data showing that host specificity may be accompanied by relatively little DNA sequence divergence. This discovery poses a problem because host specificity has been the most clear and compelling discreet phenotypic characteristic for recognizing clades in this genus. This problem, however, does not apply to the best studied Pnewnocystis organisms, which are extremely divergent at the DNA level. It is still appropriate to proceed with renaming them. It is hoped that this article will be useful to those who will undertake this task.

Interphase chromosome folding determines spatial proximity of genes participating in carcinogenic RET/PTC rearrangements.

Recurrent chromosomal rearrangements are common in cancer cells and may be influenced by nonrandom positioning of recombination-prone genetic loci in the nucleus. However, the mechanism responsible for spatial proximity of specific loci is unknown. In this study, we use an 18 Mb region on 10q11.2–21 containing the RET gene and its recombination partners, the H4 and NCOA4 (ELE1) genes, as a model chromosomal region frequently involved in RET/PTC rearrangements in thyroid cancer. RET/PTC is particularly common in tumors from children exposed to ionizing radiation. Using fluorescence in situ hybridization and three-dimensional microscopy, the locations of five different loci in this region were mapped in interphase nuclei of normal human thyroid cells. We show that RET and NCOA4 are much closer to each other than expected based on their genomic separation. Modeling of chromosome folding in this region suggests the presence of chromosome coiling with coils of ∼8 Mb in length, which positions the RET gene close to both, the NCOA4 and H4, loci. There was no significant variation in gene proximity between adult and pediatric thyroid cells. This study provides evidence for large-scale chromosome folding of the 10q11.2–21 region that offers a structural basis for nonrandom positioning and spatial proximity of potentially recombinogenic intrachromosomal loci.

Molecular and phenotypic description of Pneumocystis wakefieldiae sp. nov., a new species in rats

Organisms in the genus Pneumocystis are fungi that reside in the lungs of mammals that can cause a lethal pneumonia once the hosts lose immune function. The genus Pneumocystis contains many members, but only two species have been described formally to date, P. carinii, the type species found in rats, and P. jirovecii, resident in human beings. Rats have been shown to harbor another organism in addition to P. carinii, Pneumocystis wakefieldiae sp. nov., formerly known as Pneumocystis carinii f. sp. ratti, which is described here. Although often found together and morphologically similar, P. carinii and P. wakefieldiae are phenotypically and genetically divergent. We used the phylogenetic species recognition approach to distinguish these organisms as two distinct species and estimated the evolutionary time of their separation. Nucleotide sequence comparisons of seven homologous genes showed 4–7% divergence between the P. wakefieldiae and P. carinii sequences, which was in contrast to the 0–0.8% divergence observed within P. carinii species. Even greater divergence (30%) occurred in sequences located between genes. The MSG (major surface glycoprotein) gene families of P. carinii and P. wakefieldiae are 35% divergent from one another and differ with respect to sequence elements associated with regulation of their transcription. Differences in reactivity of monoclonal antibodies and polyclonal antisera reflected these genetically distinct surface antigens. Karyotypic analysis of P. wakefieldiae produced a single profile that was distinct from all 12 profiles known for P. carinii. Eight homologous genes were localized to chromosomes of different sizes in the two species. The cumulative genotypic and phenotypic data support a species distinction between these two organisms.

DNA sequence homology and chromosomal deletion at a site of SV40 DNA integration

The structure of simian virus 40 (SV40) DNA insertions is different from those of retrovirus proviruses and movable genetic elements. No single DNA sequence in either the cell or the SV40 genome serves as an obligatory site of SV40 integrative recombination1–9, and SV40 DNA insertions are not bordered by the repeat structures characteristic of transposons and retrovirus proviruses10–15. Integration of SV40 could involve the matching of short stretches of homologous sequences present in the otherwise heterologous SV40 and cellular genomes. To explore this possibility, I have now sequenced a rat DNA fragment that contains an unoccupied site of SV40 DNA integration. Comparison of this sequence with that of SV40 and that at the rat-SV40 recombinant junction allowed two conclusions: first, SV40 DNA became linked to rat DNA at a point where the two genomes shared 5 base pairs (bp) of DNA sequence homology; and second, a rearrangement of the rat genome, probably a deletion of at least 3 kilobases (kb), occurred at the site of SV40 integration.

Structure and expression of a tandem gene pair in Leishmania donovani that encodes a protein structurally homologous to eucaryotic cation-transporting ATPases.

An oligonucleotide probe was used to clone a cation-transporting ATPase gene from the genome of Leishmania donovani. The nucleotide sequence of the gene contained a 2,922-base-pair open reading frame that was predicted to encode a 107,406-dalton protein composed of 974 amino acids. The predicted L. donovani protein contained all the structural and functional domains expected to be present in a cation-transporting ATPase of the aspartyl phosphate class. The nucleotide sequence encoding the ATPase gene was duplicated in tandem in the parasite genome. Partial sequenation of the second member of the tandem repeat, which lay 2 kilobase pairs downstream of the ATPase gene, indicated that it was either identical to the first gene or very closely related to it. RNA homologous to either the ATPase gene or its adjacent relative was 5 kilobases in size and was approximately equally abundant in both promastigote and amastigote forms of the organism.