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.

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.

Diverse Caenorhabditis elegans genes that are upregulated in dauer larvae also show elevated transcript levels in long-lived, aged, or starved adults.

Under adverse conditions, the nematode Caenorhabditis elegans undergoes reversible developmental arrest as dauer larvae, an alternative third larval stage adapted for dispersal and long-term survival. Following such arrest, which may exceed three times their usual life-span, worms resume development to form reproductive adults of normal subsequent longevity. Mutations of genes in the dauer-formation (daf) pathway can extend life-span two- to fourfold, even in adults that mature without diapause. To identify transcript-level changes that might contribute to extended survival, we prepared a subtractive cDNA library of messages more abundant in dauer than in non-dauer (L3) larvae. Six genes were confirmed as three- to ninefold upregulated in dauer larvae, after correction for mRNA load: genes encoding poly(A)-binding protein (PABP), heat-shock proteins hsp70 and hsp90, and three novel genes of uncertain function. The novel genes encode a partial homologue of human activating signal cointegrator 1 (ASC-1), a GTP-binding homologue of a ribosomal protein, and an SH3-domain protein. Transcript levels for all except hsp70 increased during aging in two C. elegans strains, whereas the three novel genes (and possibly PABP) were also induced to varying degrees by starvation of adults. All six genes are expressed at higher levels in young adults of long-lived daf mutant strains than in normal-longevity controls, suggesting that increased expression of these genes may play a protective function, thus favoring survival in diverse contexts.

Evaluation of a single step extraction procedure for enzymatic assay of levamisole in calf serum.

A single step extraction procedure for recovery of the anthelmintic drug levamisole (LEV) from aqueous solutions and calf serum was evaluated. Levamisole was extracted from aqueous solutions and calf serum with 1.5 ml of ethyl acetate. After evaporation to dryness, the LEV content of extracts was estimated by measuring LEV inhibition of bovine milk fat globule membrane alkaline phosphatase. Lipid interference with absorbance readings was eliminated by the addition of 1.0 ml of chloroform to the assay mixtures. The recovery of LEV from both aqueous samples and serum samples by this single step extraction procedure coupled with enzymatic assay was 90%. The effective range for serum LEV determination was 0.3 to 20 micrograms/ml.