Biomedical subjects

R J Shmookler Reis

Publications and source records attributed to R J Shmookler Reis.

At least 19 recordsLinked to original sources

PubMed2001

Genetic mapping of quantitative trait loci governing longevity of Caenorhabditis elegans in recombinant-inbred progeny of a Bergerac-BO x RC301 interstrain cross.

Recombinant-inbred populations, generated from a cross between Caenorhabditis elegans strains Bergerac-BO and RC301, were used to identify quantitative trait loci (QTL) affecting nematode longevity. Genotypes of young controls and longevity-selected worms (the last-surviving 1% from a synchronously aged population) were assessed at dimorphic transposon-specific markers by multiplex polymerase chain reaction. The power of genetic mapping was enhanced, in a novel experimental design, through map expansion by accrual of recombinations over several generations, internally controlled longevity selection from a genetically heterogeneous, homozygous population, and selective genotyping of extremely long-lived worms. Analysis of individual markers indicated seven life-span QTL, situated near markers on chromosomes I (tcbn2), III (stP127), IV (stP13), V (stP6, stP23, and stP128), and X (stP41). These loci were corroborated, and mapped with increased precision, by nonparametric interval mapping-which supported all loci implicated by single-marker analysis. In addition, a life-span QTL on chromosome II (stP100-stP196), was significant only by interval mapping. Congenic lines were constructed for the longevity QTL on chromosomes III and X, by backcrossing the Bergerac-BO QTL allele into an RC301 background with selection for flanking markers. Survival data for these lines demonstrated consistent and significant effects of each QTL on life span.

Alleles↗

PubMed2001

Expression of AAV Rep proteins in SV40-transformed and untransformed cells: reciprocal interaction with host DNA synthesis.

Adeno-associated virus (AAV) inhibits the induction of host DNA synthesis by simian virus 40 (SV40) large-tumour (T) antigen, mediated through AAV-encoded 'Rep' regulatory proteins. Rep proteins are normally synthesized by AAV-infected cells only in the presence of adenovirus. However, we observed a low level of Rep protein expression in SV40 transformed cells even in the absence of helper virus. In an effort to understand the functional interaction between SV40 T antigen and regulators of AAV rep expression, we evaluated Rep protein production by cell lines transformed with various T antigen mutants known to vary in their induction of host DNA synthesis. We observed Rep protein expression proportional to SV40-induced host DNA synthesis, as measured previously for these T antigen mutants in the absence of AAV, suggesting that rep gene expression - although it opposes the oncogenic stimulation of cell cycling by SV40 - may itself be elicited by host DNA synthesis. To test this, we employed two inhibitors of DNA synthesis: hydroxyurea, which acts by depleting deoxyribose nucleotide triphosphate pools, and aphidicolin, a specific inhibitor of DNA polymerases alpha and delta. Each inhibitor markedly and significantly reduced Rep protein levels, both in immortal cells transformed by wild-type T antigen and in normal human fibroblasts, confirming the dependence of Rep protein expression on host DNA synthesis.

Antigens, Viral, Tumor↗

PubMed2000

Discrimination of primer 3'-nucleotide mismatch by taq DNA polymerase during polymerase chain reaction.

We investigated the effect of primer-template mismatch on the efficiency of polymerase chain reaction. For primers with T, C, or G as the 3' nucleotide, Thermus aquaticus (Taq) DNA polymerase was highly specific for template complementarity to this base, but was somewhat less constrained opposite the penultimate nucleotide. In contrast, primers with a 3'-terminal A were less efficiently amplified regardless of the corresponding nucleotide on the template strand. Thus, allele-specific PCR with Taq polymerase offers the greatest template discrimination (40- to 100-fold) against mismatch to a primer's 3'-terminal T, G, or C, but not A. Nucleotides at the penultimate position are responsible for roughly one-fifth as much mismatch discrimination (8- to 20-fold), and amplification efficiency is reduced when T and especially A occupy this primer position. We thus have defined conditions which allow robust discrimination for PCR-mediated analysis of single-nucleotide polymorphisms (SNPs), and for reduction in complexity of anchor-ligation PCR products.

Animals↗

PubMed2000

Anchor polymerase chain reaction display: a high-throughput method to resolve, score, and isolate dimorphic genetic markers based on interspersed repetitive DNA elements.

Genes which confer a disease when mutated, or for which population variability contributes to a quantitative trait such as longevity or disease susceptibility, can be localized in the genetic map by use of an appropriately dense set of polymorphic DNA markers. Here we describe an anchor PCR method for high-throughput genotyping, which can be used to amplify the DNA segments flanking an interspersed repetitive sequence such as a transposon, and to limit the number of product bands per reaction to facilitate marker resolution. We used this method to amplify and display DNA fragments flanking the Tc1 transposable elements from different strains of the nematode Caenorhabditis elegans, varying widely in insert number, and to analyze marker segregation in recombinant inbred lines generated from an interstrain cross. Since essentially all eukaryotic genomes contain abundant interspersed repeat families, many of which are dimorphic (for presence or absence of specific elements) among populations, this method can be used for rapid genotyping and fine-scale chromosomal mapping in many species, including those for which extensive mapping and sequencing data do not yet exist.

Animals↗

PubMed2000

Chromosomal mapping of osteopenia-associated quantitative trait loci using closely related mouse strains.

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age-dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39-40% of 227 highly-polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X-ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone-density QTL, while 3 substantiate QTLs suggested by a low-power study of 24 recombinant-inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly-related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.

Absorptiometry, Photon↗

PubMed1999

Telomerase inhibition by peptide nucleic acids reverses 'immortality' of transformed human cells.

Telomerase activity, the ability to add telomeric repeats to the ends of chromosomes, has been detected in most immortal cell lines including tumor cells, but is low or absent in most diploid, mortal cells such as those of somatic tissues. Peptide nucleic acids (PNAs), analogs of DNA or RNA which bind to complementary nucleic acids with very high affinity, were co-electroporated into immortal human cells along with a selectable plasmid. Introduction of PNAs inverse-complementary to telomerase RNA effectively inhibited telomerase activity in intact cells, shortened telomeres, reduced colony size, and arrested cell proliferation after a lag period of 5-30 cell generations, consistent with suppression of their 'immortality'. Electroporation of selection plasmid alone had no effect, while PNAs of altered sequence were markedly less effective in each assay. This constitutes the first demonstration of cell growth arrest through telomerase inhibition, upon treatment of intact cells with an exogenous compound which can be efficiently delivered in vivo. The phenotype of telomerase-inhibited transformed cells differs from senescence of normal diploid fibroblasts, but rather resembles the crisis state of incompletely transformed cells.

Amino Acid Metabolism, Inborn Errors↗

PubMed1997

Expression of SV40 large T antigen stimulates reversion of a chromosomal gene duplication in human cells.

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare "immortalization"-clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finn et al. (1989) Mol. Cell. Biol. 9, 4009-4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexisting HPRT+ cells, were confirmed as HPRT+ by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicated HPRT region, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion of HPRT mRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion to HPRT+ was unaltered during the normal in vitro lifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; each P < 10(-5)). Stimulation of HPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10-30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.

Antigens, Polyomavirus Transforming↗

PubMed1997

Quantitative trait loci controlling halothane sensitivity in Caenorhabditis elegans.

Genetic analysis is an essential tool for defining the molecular mechanisms whereby volatile anesthetics (VA) disrupt nervous system function. However, the degree of natural variation of the genetic determinants of VA sensitivity has not been determined nor have mutagenesis approaches been very successful at isolating significantly resistant mutant strains. Thus, a quantitative genetic approach was taken toward these goals. Recombinant-inbred strains derived from two evolutionarily distinct lineages of the nematode Caenorhabditis elegans were tested for sensitivity to clinically relevant concentrations (0.3-0.5 mM) of the VA halothane. The halothane sensitivities of coordinated movement and male mating behavior were highly variant among the recombinant-inbred strains with a range of EC50 values of 13- and 4-fold, respectively. Both traits were highly heritable (H2 = 0.82, 0.87, respectively). Several strains were found to be significantly resistant to halothane when compared with the wild-type strain N2. A major locus or loci mapping to the middle of chromosome V accounted for more than 40% of the phenotypic variance for both traits. Five weaker loci, four of which interact, explained most of the remaining variance. None of the halothane-sensitivity quantitative trait loci significantly affected behavior in the absence of halothane or halothane's potency for C. elegans immobilization, which requires 5-fold higher drug concentrations. Thus, the quantitative trait loci are unlikely to result from differences in halothane-independent (native) behavior or differences in halothane metabolism or permeability. Rather, these loci may code for targets and/or downstream effectors of halothane in the C. elegans nervous system or for modifiers of such gene products.

Anesthetics, Inhalation↗

PubMed1997

Carcinogens stimulate intrachromosomal homologous recombination at an endogenous locus in human diploid fibroblasts.

Mitotic recombination is believed to play an important role in the development of many cancers. An improved system has been developed to detect reversion of an intragenic DNA duplication, as a model for intrachromosomal homologous recombination. The 'LNtd' strain of human fibroblasts, derived from a Lesch-Nyhan donor, produces no detectable hypoxanthine phosphoribosyltransferase (HPRT) activity due to a 13.7-kilobase-pair DNA insertion duplicating exons 2 and 3 of the HPRT locus. These cells are therefore sensitive to selection in HAT medium, against cells lacking functional HPRT enzyme. Clonal reversion to HAT resistance occurs spontaneously at 1-3 x 10(-5)/cell/generation, and can be induced by brief exposure to a variety of carcinogenic agents. Six known carcinogens, including two (diethylstilbestrol and nickel chloride) which were non-mutagenic in Salmonella by Ames HIS-reversion tests, showed dose-dependent induction of LNtd reversion by a maximum of 2.4- to > 11-fold over controls (each p < 0.01). In contrast, 5 non-carcinogenic agents, including two 'Ames-positive' chemicals, sodium azide and 8-hydroxyquinoline, evoked no more than a 1.7-fold increase in reversion (not significant). The molecular events associated with reversion to HAT-resistance were characterized, relative to the parental strain, in HATR clones derived from either untreated or carcinogen-treated cells. Both the intron-3:intron-1 junction situated between the duplicated HPRT segments in LNtd cells (amplified by polymerase chain reaction), and a restriction fragment corresponding to the duplicated HPRT DNA (assessed by Southern-blot hybridization), were lost from the majority of HATR revertant clones, whether they arose spontaneously or following exposure to Cr(VI) or ultraviolet light. These results imply that HATR reversion is induced in LNtd cells by carcinogenic treatments, through a mechanism consistent with homologous recombination, and is highly concordant with induction of in vivo carcinogenesis by the same agents.

Aminopterin↗

PubMed1997

Role of transposable elements in age-related genomic instability.

Genetic instability is associated with aging in many species. One of the initiating factors for genetic instability is the movement of transposable elements (TEs), which occur in all prokaryotic and eukaryotic organisms. The hypothesis that TEs could be involved in the aging process is discussed and the correlation between aging and activity of TEs is analysed in a variety of biological systems.

Age Factors↗

PubMed1997

Induction of duplication reversion in human fibroblasts, by wild-type and mutated SV40 T antigen, covaries with the ability to induce host DNA synthesis.

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.

Antigens, Polyomavirus Transforming↗

PubMed1997

Cellular and molecular biomarkers indicate precocious in vitro senescence in fibroblasts from SAMP6 mice. Evidence supporting a murine model of premature senescence and osteopenia.

A variety of short-lived mouse strains (SAMP strains) and control strains of less abbreviated life span (SAMR strains) have been proposed as murine models of accelerated senescence. Each SAMP strain, in addition to displaying "progeroid" traits of accelerated aging, exhibits a singular age-related pathology. The application of this animal model to the study of normal aging processes has been and remains controversial. Therefore, we have undertaken a study of dermal fibroblasts derived from the short-lived SAMP6 strain, which shows early-onset and progressive osteopenia. We have investigated cellular and molecular characteristics that are associated with in vitro aging of normal human fibroblasts, and which are exacerbated in fibroblasts from patients with Werner syndrome, a human model of premature senescence. We found that SAMP6 dermal fibroblasts, relative to SAMR1 and C57BL/6 controls, exhibit characteristics of premature or accelerated cellular senescence with regard to in vitro life span, initial growth rate, and patterns of gene expression.

Aging, Premature↗

PubMed1997

Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase.

Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two- to fivefold. T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.

Antigens, Polyomavirus Transforming↗

PubMed1996

Reduced telomere length in ataxia-telangiectasia fibroblasts.

Chromosomal instability with a high frequency of telomere fusion is characteristic of ataxia-telangiectasia cells both in vivo and in vitro. We have measured telomere length and found it to be consistently reduced in both diploid and SV40-transformed cells A-T fibroblasts, relative to control cells. We examined a few possible mechanisms which might account for telomeric length reduction, including telomerase activity in transformed cells and endogenous nuclease activities, but found no differences between A-T and control cells in these parameters.

Ataxia Telangiectasia↗

PubMed1996

Defining genes that govern longevity in Caenorhabditis elegans.

We previously identified five regions on the chromosomal map of Caenorhabditis elegans, containing genes that help specify life span in this species, by comparing the genotypes of young and long-lived progeny from a cross between strains Bristol-N2 and Bergerac-BO [Ebert et al. (1993): Genetics 135:1003-1010]. Analyses of additional crosses, and of putative polymorphisms for the implicated genes, are necessary to clarify the roles of naturally occurring polymorphic alleles in determining longevity. We therefore carried out a second multigenerational cross, between strains Bristol-N2 and DH424 (both nonmutators at 20 degrees C), to create a different heterogeneous recombinant-inbred population. We again found strong evidence implicating multiple genes, which differ between the parental strains, in the determination of life span. Increased variance of survival, for F2 and homozygous F25 worms relative to F1 hybrids, is consistent with such alleles assorting randomly in the cross progeny. Moreover, chromosome mapping data corroborate the polygenic nature of this quantitative trait. Genotypes of young and very long-lived adult worms from a synchronous F15 population were determined by polymerase chain reaction, to identify the parental strain of origin for each of 10 polymorphic loci. Two regions, on chromosomes II and IV, each contain at least one gene with allelic differences in associated longevity. A recombinant-inbred Bergerac-BO x Bristol-N2 population, derived from the earlier cross between those strains, was exposed to an acute toxic level of hydrogen peroxide. Genotyping of H2O2-resistant worms implicated at least one of the five chromosomal regions previously identified in the same cross progeny as harboring a longevity-determining gene. Superoxide dismutase and catalase levels, determined for the three parental strains as they aged, confirm the existence of polymorphisms in the corresponding genes (or their regulatory mechanisms) inferred from the chromosome-II mapping data, and are consistent with the hypothesis that increased longevity is conferred by high levels of these enzymes late in life.

Age Factors↗

PubMed1995

Strain evolution in Caenorhabditis elegans: transposable elements as markers of interstrain evolutionary history.

Evolutionary relationships across taxa can be deduced from sequence divergence of proteins, RNA, or DNA; sequences which diverge rapidly, such as those of mitochondrial genes, have been especially useful for comparisons of closely related species, and--within limits--of strains within a species. We have utilized the transposable element Tc1 as a polymorphic marker to evaluate the evolutionary relationships among nine Caenorhabditis elegans strains. For five low-Tc1-copy strains, we compared patterns of restriction fragments hybridizing to a cloned Tc1 probe. Twenty of the 40 Tc1 insertion sites thus characterized were common to all five strains, and so presumably preceded strain divergence; the 20 differential bands were used to construct a maximum-parsimony tree relating these strains. In four high-copy-number stocks (three wild-type strains and a subline), we determined occupancy of 35 individual Tc1 insertion sites by a polymerase chain reaction assay. Surprisingly, the high-copy strains share a common subset of these Tc1 insertions, and the chromosomal distribution of conserved Tc1 sites is "clustered" with respect to the other elements tested. These data imply a close evolutionary relationship among the high-copy strains, such that two of these strains appear to have been derived from the highest-copy-number lineage (represented by two stocks) through crossing with a low-Tc1 strain. Abundances of Tc1 elements were also estimated for the four high-copy-number stocks, at approximately 200-500 copies per haploid genome, by quantitative dot-blot hybridization relative to two low-copy strains. Annealing with 32P-labeled probes corresponding to full-length Tc1, an oligonucleotide within the Tc1 terminal inverted repeats, and an internal Tc1 oligonucleotide, gave essentially identical results--indicating that Tc1 termini exist in the genome primarily as components of full-length Tc1 elements. A composite evolutionary tree is proposed, based on the locations and numbers of Tc1 elements in these strains, which is consistent with a four-branch intraspecific tree deduced previously by maximum-parsimony analyses of mitochondrial sequence changes; it also serves to elucidate the evolutionary history of transposon mobility.

Animals↗

PubMed1994

Age-dependent somatic excision of transposable element Tc1 in Caenorhabditis elegans.

The Tc1 element of the free-living nematode Caenorhabditis elegans is a well characterized transposon that is present in 30-500 copies per haploid genome, depending on the strain. Excision of Tc1 elements, which occurs readily in somatic tissues during larval development, has not previously been examined during aging of adult worms. We have identified a recently inserted Tc1 element in the KR1787 mutator strain of C. elegans and have found that Tc1 somatic excision at that site increases by more than 14-fold during the organism's lifespan.

Aging↗

PubMed1993

Longevity-determining genes in Caenorhabditis elegans: chromosomal mapping of multiple noninteractive loci.

We have used chromosome mapping with polymorphic markers to define genetic components governing life span in the nematode Caenorhabditis elegans. A complex recombinant-inbred population was derived from an interstrain cross, yielding > 1000 genotypes, each a composite of homozygous segments from the two parental strains. Genotypes were analyzed for the last-surviving 1-5% of worms in aging cohorts, and for young controls, by multiplex polymerase chain reaction using polymorphic markers to distinguish the parental alleles. We identified five regions of the genome at which one parental allele was significantly enriched in long-lived subpopulations. At four of five loci, the same alleles were selected in aging cohorts maintained under two different conditions, implying that these genes determine life span in differing environments.

Animals↗