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.

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.

Mutation induction in Escherichia coli WP2 uvrA by Cerenkov emission associated with 137Cs gamma irradiation.

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs gamma irradiation. Furthermore, the data strongly suggest a more than additive interaction between the gamma-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/10(7) survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by gamma irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.

Ammonia, respiration, and longevity in nematodes: insights on metabolic regulation of life span from temporal rescaling.

To better understand metabolic correlates of longevity, we used a graphical technique to compare the adult temporal patterns of several markers of metabolic activity - ammonia elimination, oxygen consumption rate, ATP levels, and (in freeze-permeabilized worms) the rate of NADPH-activated, lucigenin-mediated superoxide formation - as observed by us and others in normal and long-lived mutant Caenorhabditis elegans strains. All of these traits declined with time, and when their logarithms were plotted against time, appeared reasonably linear for most of the duration of the experiments. The profiles for ammonia output conformed well to parallel regression lines; those for the other metabolic parameters varied widely in slope as originally plotted by the authors, but much less so when replotted as logarithms against adjusted time, scaled by the reciprocal of strain longevity. This is consistent with coregulation of life span, respiration rate, ATP levels, lucigenin reactivity, but not ammonia excretion, by a physiological clock distinguishable from chronologic time. Plots, scaled appropriately for equalized slopes, highlighted y-axis intercept differences among strains. On rescaled plots, these constitute deviations from the expectation based on 'strain-specific clock' differences alone. With one exception, y-intercept effects were observed only for mutants in an insulin-like signaling pathway.

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.

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.