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J R Stringer

Publications and source records attributed to J R Stringer.

At least 19 recordsLinked to original sources
PubMed2006

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 approximately 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.

Adolescent↗
PubMed2004

Expression and complexity of the PRT1 multigene family of Pneumocystis carinii.

Pneumocystis carinii has a multigene family, PRT1, that encodes proteins with homology to KEX2-like proteases. PRT1 genes cluster with MSG genes near the telomeres and, like MSG, PRT1 proteins seem to be surface-expressed. The clustering of PRT1 and MSG genes suggested that expression of the two multigene families might be coordinated. Studying gene expression in P. carinii has been hampered by the lack of a culture system, and by lack of clonality in P. carinii populations in naturally infected rats, the host of this fungus. Heterogeneity can be reduced, however, by low-dose intratracheal inoculation, which can produce P. carinii populations dominated by organisms derived from a single progenitor. To study PRT1 expression, nude rats were inoculated with approximately 10 P. carinii each. The clonality of the P. carinii populations from inoculated rats was assessed by analysis of the UCS locus, a site in the genome that is known to be very heterogeneous in naturally infected rats, but nearly homogeneous in rats infected by low-dose intratracheal inoculation. Each of the populations had the same MSG gene at the UCS locus in at least 80 % of the organisms. To investigate PRT1 gene expression, RNA was amplified using primers that amplify numerous PRT1 genes. Seventy-four cloned cDNAs were sequenced, including at least 12 clones from each population of P. carinii. Many differently expressed PRT1 sequences were identified in each population, and a total of 45 different sequences were detected. However, the same PRT1 sequence was present in 15 of 74 plasmids and was found in 3 of the 5 P. carinii populations, suggesting that some PRT1 genes may be either more commonly expressed or expressed at a higher level. These data show that many members of the PRT1 gene family can be expressed in populations of P. carinii derived from few progenitors and suggest that the regulation of this family is different from that governing expression of the MSG gene family.

Animals↗
PubMed2001

Time between inoculations and karyotype forms of Pneumocystis carinii f. sp. Carinii influence outcome of experimental coinfections in rats.

The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.

Adenosine Triphosphate↗
PubMed2001

Genetics of surface antigen expression in Pneumocystis carinii.

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 protozoan Trypanosoma brucei and the bacteria Borrelia spp.

Borrelia↗
PubMed2001

The relation between peripheral blood leukocyte counts and respiratory symptoms, atopy, lung function, and airway responsiveness in adults.

STUDY OBJECTIVES: Eosinophils and neutrophils play major roles, respectively, in the pathogenesis of asthma and COPD, and it is well recognized that levels of these cells in peripheral blood are increased in relation to their pulmonary involvement. However, the relation between peripheral blood cell counts of the other major leukocyte groups and these lung diseases or markers of allergy or airflow obstruction is less clear. We have therefore investigated the association between peripheral blood levels of eosinophils, neutrophils, basophils, monocytes, and lymphocytes and the occurrence of chronic respiratory symptoms, atopy, lung function, and bronchial hyperresponsiveness, and the modifying effect of age, in adults. DESIGN: A cross-sectional general population study. SETTING: Data on > 2,000 British adults, who originally participated in a study of diet and lung health, were analyzed using multiple linear and logistic regression to adjust for potential confounders, including age, sex, and smoking history. RESULTS: We found that, like eosinophils, the peripheral basophil count was increased in relation to asthma and associated symptoms, and to airway hyperreactivity and increased total IgE, but differed from eosinophils in that basophils were unrelated to atopy. Monocytes were predominantly associated with symptoms indicative of obstructive airway disease, in similar relation to neutrophils, but both of these leukocyte counts were also increased in asthma patients in older age groups. Lymphocyte counts were unrelated to any objective or subjective marker of disease. CONCLUSIONS: If peripheral blood cell counts reflect pulmonary involvement of these leukocyte groups, basophils and monocytes may play a distinct role in the pathogenesis of allergic and nonallergic respiratory disease.

Adolescent↗
PubMed2000

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

Adult↗
PubMed2000

The major surface glycoprotein expression sites of two special forms of rat Pneumocystis carinii differ in structure.

Pneumocystis carinii forma specialis carinii has a unique locus called the UCS, which controls surface antigen variation by acting as the expression site for a family of genes encoding isoforms of the major surface glycoprotein (MSG). Every MSG mRNA begins with 380 nucleotides copied from the UCS locus. This UCS leader sequence is necessary for initiation of translation and for protein processing. Given the UCS's importance in P. carinii f. sp. carinii, it was of interest to examine the UCS locus in the related P. carinii f. sp. ratti. The first 380 nucleotides of P. carinii f. sp. ratti MSG mRNAs were 59% identical to the P. carinii f. sp. carinii UCS. However, the DNA encoding the P. carinii f. sp. ratti UCS was not unique in the genome. Instead, only the first 97 nucleotides came from a unique locus and the remainder from >/=7 loci. These data suggest that the two organisms may use different mechanisms to generate surface variation.

Animals↗
PubMed2000

An analysis of technology usage for streaming digital video in support of a preclinical curriculum.

Usage of streaming digital video of lectures in preclinical courses was measured by analysis of the data in the log file maintained on the web server. We observed that students use the video when it is available. They do not use it to replace classroom attendance but rather for review before examinations or when a class has been missed. Usage of video has not increased significantly for any course within the 18 month duration of this project.

California↗
PubMed1999

Major surface glycoprotein genes from Pneumocystis carinii f. sp. ratti.

Pneumocystis carinii occurs in a variety of mammals, each of which harbors one or more genetically distinct "special forms" of the microbe. Laboratory rats can be infected by two special forms, P. carinii f. sp. ratti and P. carinii f. sp. carinii. P. carinii f. sp. carinii has a variable antigen, the major surface glycoprotein (MSG), the expression of which is controlled by genetic recombination. Recombination may involve the CRJE, a 23-bp DNA sequence element invariant among P. carinii f. sp. carinii MSG genes. To better understand the role of the CRJE in MSG gene expression and to explore the possible role of MSG in P. carinii infection in rats, P. carinii f. sp. ratti MSG genes were studied. These genes were found to be related to MSG genes of P. carinii f. sp. carinii, but less so than MSG genes from P. carinii f. sp. carinii are to each other. P. carinii f. sp. ratti MSG genes were present throughout the genome and were expressed as an abundant mRNA species slightly smaller than that found in P. carinii f. sp. carinii. P. carinii f. sp. ratti MSG transcripts included a CRJE-like sequence only 78% identical to the CRJE of P. carinii f. sp. carinii. Comparison of MSG proteins from the two rat special forms of P. carinii to those from human, ferret, and mouse P. carinii did not support the hypothesis that growth in the rat lung requires certain primary MSG peptide sequences.

Amino Acid Sequence↗
PubMed1999

A new family of Pneumocystis carinii genes related to those encoding the major surface glycoprotein.

A new family of Pneumocystis carinii genes (called MSR for MSG-related) that encodes peptides related to the major surface glycoprotein (MSG) is described. Members of the MSR sequence family are linked to MSG genes and are located near the ends of at least 13 chromosomes. Transcripts encoding different MSR isoforms were present in a single population of P. carinii f. sp. carinii, showing that multiple MSR genes were expressed. Two size classes of MSR mRNA, 2.4 and 3.5 kb, were detected. Both sizes of MSR mRNA lacked the upstream conserved sequence (UCS), which is found on the 5' end of MSG mRNAs because MSG genes must be linked to the UCS to be transcribed. The absence of the UCS from MSR mRNAs suggests that expression of MSR genes does not require linkage to the UCS locus.

5' Untranslated Regions↗
PubMed1998

In situ detection of frameshift mutations in mouse cells.

A reporter gene system that allows in situ detection of cells that have suffered a specific frameshift mutation was developed. To construct the reporter gene, the open reading frame of a human placental alkaline phosphatase (PLAP) gene was disrupted by insertion of either 5 or 7 G:C basepairs, which formed mutant alleles carrying 9 and 11 consecutive G residues, respectively. The mutant PLAP genes did not produce alkaline phosphatase activity in cultured mouse cells in transient transfection assays. Several cell lines that contained integrated copies of the mutant PLAP genes were made. Histochemical staining of fixed cells showed that these cell lines contained a small number of cells that expressed PLAP activity and bound antibodies directed against PLAP. Cells carrying the allele with 11 consecutive G residues (G11 allele) acquired PLAP activity at a rate between 2 x 10(-3) and 2 x 10(-4) events per cell per generation, depending on the cell line. Cells carrying the allele with 9 consecutive G residues (G09 allele) acquired PLAP activity at a rate between 2 x 10(-5) and 2 x 10(-6) events per cell per generation, depending on the cell line. Cultures of PLAP+ cells were derived from cell lines carrying PLAP mutant genes. All the cells in these cultures had PLAP activity and bound anti-PLAP antibody. PLAP mRNA levels were the same in cultures where all cells were PLAP+ and in cultures where less than 1% of the cells expressed PLAP activity. DNA sequence analysis of PLAP+ cells showed that the G11 allele reverted by losing one basepair, and the G09 allele reverted by gaining one basepair.

Alkaline Phosphatase↗
PubMed1998

Use of human placental alkaline phosphatase transgenes to detect somatic mutation in mice in situ.

Methods for in situ detection of cells that have suffered a specific mutation would be valuable for understanding somatic genetic mosaicism, a phenomenon that underlies a variety of diseases including cancer. Such methods would also be valuable in studying changes in gene expression, whether programmed by the cells or caused by exogenous forces, such as exposure to genotoxins or infection by a virus. To improve methods for detection of genetic change at the cellular level in animal tissues, we used the human placental alkaline phosphatase (PLAP) gene. The PLAP gene sequence was modified such that it could no longer produce functional PLAP enzyme. Mutant PLAP genes were placed in the mouse genome, and populations of cells carrying these mutant PLAP genes were studied to determine the fraction of cells that would acquire PLAP activity. Spontaneous and induced reversion of mutant PLAP genes was studied in cultured cells and in the tissues of transgenic mice. The data obtained from these studies show the utility of in situ reporter genes such as PLAP for detection of variant cells within a tissue.

3T3 Cells↗
PubMed1998

The genome of Pneumocystis carinii.

The best understood special form of P. carinii, P. carinii formae specialis (f.sp.) carinii, appears to be haploid and contains about 8 million base pairs of DNA (8.5 fg) per nucleus. The genome of P. carinii f.sp. carinii is divided into 13-15 linear chromosomes that range from 300 to 700 kb in size. Eight different P. carinii f.sp. carinii karyotypes have been observed. The karyotypes of P. carinii f.sp. carinii differ due to slight variations in the lengths of chromosomes, but the 8 karyotype-forms of P. carinii f.sp. carinii exhibit very little variation in DNA sequence. By contrast, the genome of P. carinii f.sp. carinii differs markedly in sequence from the genomes of P. carinii from other hosts, such as mouse, ferret and human. In addition, chromosomes and DNA sequences from P. carinii from mouse, ferret, and human also differ greatly from each other. The genome of a ferret P. carinii appears to be up to 1.7 times larger than those of P. carinii from other hosts. Nearly two dozen P. carinii genes have been cloned and sequenced. The typical P. carinii gene sequence is 60-65% A+T. P. carinii genes usually contain introns, which are typically less than 50 bp in length, but can be as numerous as 9 per gene. A system for naming P. carinii genes is proposed in which each gene would be designated by an italic three-letter lower case symbol. The first allele (i.e. sequence) that is found would have a superscript 1, such as xyz1(1). Any subsequent alleles would be designated as xyz1(2), etc. A protein would have the same symbol as the gene that produced it, but written in roman print with the first letter an uppercase, such as Msg1. Some of the P. carinii genome is comprised of DNA sequences that are present dozens of times. Three families of such repeated DNA sequences have been described. Two of these families (MSG and PRT) encode proteins. The third family is the telomere repeat, which is found at the ends of each chromosome, and sometimes at internal chromosomal sites, in which case it has been called the alpha repeat. Determination of the complete sequence of the P. carinii genome is both practicable and of primary importance to the understanding of this organism. The small size of the P. carinii genome and its packaging into chromosomes that are resolvable by PFGE will facilitate sequence analysis.

Chromosomes, Fungal↗
PubMed1998

Expression, structure, and location of epitopes of the major surface glycoprotein of Pneumocystis carinii f. sp. carinii.

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5' end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein-MSG-B fusion protein ([MBP]MSG-B41-1065) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

Animals↗
PubMed1998

Identification of a putative precursor to the major surface glycoprotein of Pneumocystis carinii.

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5' end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.

Amino Acid Sequence↗