Srinivas Ayyadevara et al. (2003) International Worm Meeting "Fine Mapping of Quantitative Trait Loci Conferring Resistance to Heat-Shock and Ultraviolet Radiation in C. elegans"

Srinivas Ayyadevara, Ping Kang, Robert Shmookler Reis

[International Worm Meeting, 2003]

We used a novel Tc1-based fine mapping procedure and recombinant inbred (RI) lines to map Quantitative Trait loci (QTLs) for resistance to heat shock and ultraviolet radiation in Caenorhabditis elegans. RI lines, generated from a Bristol-N2 Bergerac-BO cross, were exposed to UV light or heated to 35C, and subsequent survival measured. The RI lines differed markedly with respect to resistance to either stress. Linkage of stress-resistance QTLs to Tc1-based markers was determined by composite interval mapping (Zeng 1994). Quantitative trait loci significantly affecting resistance to UV were positioned on chromosomes I, II and III, with peak LOD scores of 3.3, 9.1 and 3.5, respectively. We found three significant QTLs for heat shock resistance on chromosome IV, and one such QTL on the X chromosome. Two of these loci, one in the middle of chromosome IV (near stP35) and one near the left end of X (between stP41 and stP40) were close or identical to QTLs affecting life span, as determined in an earlier mapping study of recombinant inbred populations (Ayyadevara et al. 2001). All significance thresholds shown (horizontal lines in Figure) were 0.05 genome-wide false positive levels determined empirically by 1000 permutations of genotype with respect to phenotype for each trait mapping (Churchill and Doerge, 1994).

Shook DR et al. (1996) West Coast Worm Meeting "QTLS FOR LIFE HISTORY TRAITS IN CAENORHABDITIS ELEGANS"

Hinerfeld DA, Shook DR, Johnson TE

[West Coast Worm Meeting, 1996]

The evolution of genes affecting senescence has been hypothesized to depend on the other life history traits (traits affecting survival or fertility). Hence we have used an approach which allows us not only to identify individual loci specifying various life history traits, but also allows us to detect interactions among life history traits at individual loci and interactions between loci for individual traits. 81 recombinant inbred (RI) strains generated from two wild type parental strains, Bergerac (BO) by Bristol (N2), were used. Each RI strain was genotyped for 40 markers, which were based on Tc1 insertions present in one parental genome (generally BO) but not the other. Each RI strain was also assayed for several different life history traits. This data set was used to generate QTL maps for each trait, which allowed us to compare the effects of each locus on several traits. In addition, we tested for epistasis among loci for each trait. We have identified several putative quantitative trait loci (QTLs) for life history traits in self fertilizing hermaphrodites. We have found QTLs for mean and maximum life span, early, mean and maximum fertility, "bagging" (internal hatching of progeny), temperature sensitive fertility, growth rate, and age of onset of reproduction. One locus shows pleiotropic effects on the survival characters mean life span and bagging, while at least two loci show pleiotropic effects among some of the fertility characters. No loci showed pleiotropy, negative or otherwise, for both survival and fertility characters. Several pairs of loci show epistatic interactions for various life span and fertility traits.

Ebert RHA et al. (1995) International C. elegans Meeting "ANTIOXIDANT ENZYME ACTIVITIES AND MAPPING OF H2O2 RESISTANCE IN THREE STRAINS OF C. ELEGANS"

Ebert RHA, Shammas MA, Sohal BH, Sohal RS, Shmookler Reis RJ

[International C. elegans Meeting, 1995]

One factor determining the rate of senescence of an organism may be the level of oxidative stress it experiences throughout its lifespan. Recent studies of the long-lived mutant age-1 (Larsen, PNAS 90, 8905, 1993; Vanfleteren, Biochem J 292, 605, 1993) reveal that activity levels of both catalase and Cu-Zn SOD are elevated in old worms compared to controls. Our work with N2 BO (Ebert et al., Genetics 135, 1003, 1993) and N2 DH424 (submitted) recombinant-inbred (RI) populations indicates that there are genes with allelic differences between the parental strains, which have differential effects on longevity. Here we have tested whether SOD and catalase activities vary in an age- or strain- dependent manner. Middle-aged worms (~70-85% of the cohort still alive) were compared with old worms (~4-6% alive). SOD activity levels decreased with age for cohorts of strains DH424 and RW7000 (BO) by ~50%, but remained at the same level for an N2 population. Catalase activity levels dropped with increasing age for populations of all three strains tested and the decrease was largest for N2 (to 38% of the earlier value). Currently, we are determining the sensitivity to acute hydrogen peroxide (H2O2) exposure for these strains and for a cohort of N2 BO RI progeny. We are also analyzing RI survivors of acute H2O2 exposure by single-worm PCR (Williams et al., Genetics 131, 609, 1992) to map any polymorphic genes responsible for their resistance to this pro-oxidant.

Iwato, A. et al. (2019) International Worm Meeting "Identification of genes required for natural variation affecting temperature acclimation."

Ohta, A., Okahata, M., Mitani, S., Iwato, A., Kuhara, A., Koyama, H., Yoshina, S., Minakuchi, Y., Toyoda, A.

[International Worm Meeting, 2019]

Temperature is one of the critical environmental factors for creatures. We are studying natural variation in temperature acclimation of C. elegans. 15 degree-cultivated N2 can survive at 2 degree, meanwhile 25 degree cultivated N2 can not survive at 2 degree. Only 3 hours after cultivation temperature-shift from 25 to 15 degree, cold acclimation was newly established(Ohta et al., Nature common, 2014; Okahata et al., Science Advances, 2019). AB1 from Australia can rapidly acclimate to new temperature. On the other hand, CB4856 from Hawaii can acclimate slowly. To identify the gene involved in the natural variation of temperature acclimation, we decoded whole genome DNA sequences of three natural variants including AB1 by using next generation sequencer (Okahata et al., JCPB, 2016). We made recombinants inbred(RI) lines by crossing AB1 with CB4856, and isolated 71 RI lines. The gene responsible for rapid temperature acclimation in AB1 was mapped on specific region of chromosome I, by performing SNP analysis (Okahata et al., JCPB, 2016). To narrow the mapping region, we obtained more RI lines and compared their cold acclimation phenotype and genotype with next generation sequencer. The candidate genes responsible for rapidtemperature acclimation in AB1were narrowed down to 15 genes. We then measured cold acclimation of knockout mutants of them, so far, VH15N14R.1 mutant lacking a novel gene showed the abnormal cold acclimation. VH15N14R.1 protein in CB4856 strain has the substitutions of two amino acid residues and the deletion of an arginine, comparing to AB1 strain. VH15N14R.1 gene was expressed in head neurons including small pairs of sensory neurons. The expression levels of VH15N14R.1 gene was increased when animals were cultivated at 25 degree compared with animals cultivated at 15 degree. We are going to identify the cell where VH15N14R.1 regulate cold acclimation, and determine a molecular function of VH15N14R.1 protein.

S Ayyadevara et al. (1999) International C. elegans Meeting "Genetic Mapping of Multiple Loci Determining Life Span in C. elegans"

S Ayyadevara, RJ Shmookler Reis, JJ Thaden

[International C. elegans Meeting, 1999]

We conducted two new inter-strain crosses, between C. elegans strains Bergerac-BO and either CL2a or RC301, and created a heterogeneous recombinant inbred (RI) population from each, for a total of four crosses constructed and analyzed to date. The longest-lived 1% of worms in each RI population and comparable numbers of unselected young worms were genotyped. Data were then analyzed by maximum-likelihood scanning using both composite interval mapping and nonparametric interval mapping. We have now observed a total of 12 loci with highly significant LOD scores (10 in the present two crosses), each well above the p<0.05 threshold for genome-wide scans. Since genetic mapping in interstrain crosses can only detect loci which are dimorphic in the parental strains, each cross can identify a subset of the total life-span affecting loci. From the number of loci shared by multiple inter-strain crosses, we estimate that C. elegans harbors only a few dozen polymorphic genes with a comparable effect on life span to the 12 QTLs we have found. We have now initiated back-crosses to construct multiple congenic (near-isogenic) lines for each QTL region in a parent of contrasting phenotype. Several of these back-crossed lines have been tested for longevity and differ significantly from the recurrent parent. Supported by NIH grant AG-09413.

Ayyadevara S et al. (1995) International C. elegans Meeting "MAPPING OF LONGEVITY DETERMINING GENES IN C. ELEGANS"

Thaden JJ, Ayyadevara S, Ebert RHA, Shmookler Reis RJ

[International C. elegans Meeting, 1995]

Absence of inbreeding depression, short mean life span and reduced generation time make C. elegans an excellent model system for studying genes that determine longevity. Recombinant inbred strains have been used in a number of organisms to map genes of aging. Recombinant inbred (RI) lines were generated from a cross between a low-Tc1-copy strain (Cl2a) and a high-copy strain (Bergerac-BO). A similar cross (Ebert et al; Genetics 1993) was made between Bristol N2 and Bergerac-BO for the same purpose, and the genotypes were analysed by using sequence-tagged strain specific markers. As aging is polygenic and any two strains will be polymorphic at only some of the relevent loci the present cross is aimed at mapping additional genes that determine longevity. Bergerac-BO and Cl2a worms were crossed for seven generations to accumulate recombinations, followed by five generations of selffertilizations to achieve homozygsoity. Analysed genotypes of young and old worms from a synchronously aging population, will be presented.

van Swinderen B et al. (1995) International C. elegans Meeting "INTER-BEHAVIORAL QTL STUDY OF ANESTHETIC RESPONSE"

van Swinderen B, Crowder CM

[International C. elegans Meeting, 1995]

We are studying the behavior genetics of anesthesia in C. elegans by using Recombinant Inbred(RI) lines. Based on electrophysiologic studies in vertebrates, the response to anesthetics may involve several genes. Genetic polymorphisms created by mosaic Bristol (N2) and Bergerac (BO) genomes provide an alternative to classical mutagenesis for mapping such loci. Two sets of RIs were examined in a preliminary study of select behaviors (these RIs, kindly provided by Tom Johnson" !Affiliation "[1] and Robert Shmookler Reis" !Affiliation "[2], are already genotyped for Tc1 polymorphic markers). Chemotaxis and other C. elegans behaviors have been shown to be significantly more sensitive than mobility to the volatile anesthetic halothane." !Affiliation "[3] An inter-behavioral Quantitative Trait Loci (QTL) analysis of anesthetic response was initiated by generating dose-response curves for 40 RIs for chemotaxis and mobility. The RIs were polymorphic for both behaviors in halothane: EC50 values for chemotaxis derived from dose-response curves for each RI strain ranged from 0.08 to 1.82 vol % halothane, while the N2 strain had an EC50 of 0.49=B10.064. About one-fourth of the RIs were significantly more sensitive than N2, and a few were more resistant to halothane's effect on chemotaxis. The same RIs were assayed for immobility, and the EC50 range was found to be 3.0 to 5.6 vol % halothane; the EC50 for N2 is 3.58=B10.069. Surprisingly, most RIs tested for immobility were found to be more resistant than N2. Further, patterns of sensitivity and resistance to halothane in the various RIs did not necessarily correlate between the immobility and chemotaxis assays, suggesting that different QTLs may be involved in the different behavioral effects of anesthetics. Marker correlations with EC50s suggest inter-behavioral QTL overlaps on chromosomes II and III, but a mobility-specific QTL on chromosome V. Synthetic gene interactions between N2 and BO alleles appear to be an important factor in creating phenotypic variability in the RIs. 1 Behav. Gen.16(#1): 221-235 (1986). 2 Genetics 135: 1003-1010 (1993). 3 WBG 13(#3): 76 (1994).

Ayesha N Shajahan et al. (2001) International C. elegans Meeting "Regulation of endocytosis of albumin in the intestinal epithelial cells of C.elegans by TGFbeta-like signaling"

Ayesha N Shajahan, Shahid S Siddiqui

[International C. elegans Meeting, 2001]

In C. elegans , TGF b -like signaling pathways regulate a range of developmental processes such as dauer formation (Estevez et al., 1993), body size growth and male tail formation (Morita et al., 1999; Suzuki et al., 1999), and axon guidance (Colavita et al., 1998). Recently, Siddiqui et al. have shown that daf-4 (TGF b -like-RII) and daf-1 (TGF b -like-RI) (at 25 o C) mutants show poor uptake of FITC-BSA (fluorescein-5-isothiocyanate isomer- bovine serum albumin) in the intestinal cells when compared to wild-type animals. Our aim is to identify the genes in the TGF b -like pathways that affect endocytosis of albumin in the intestinal epithelial cells. We will examine the uptake of FITC-BSA in the mutants of the TGF b -like pathway in comparison to its uptake in wild type. daf-4::gfp (Patterson et al., 1997) and daf-1::gfp (Gunther et al., 2000) are expressed in the intestinal cells. Expression and regulation of other TGF b -like receptors, Smad proteins and the downstream trancription factors in the TGF b -like signaling pathway will be examined in the intestinal cells of wild type and various daf mutants, in the presence and absence of albumin, by immunofluorescence and immunoprecipitation assays. This approach will determine the role of TGF b -like signaling in endocytosis of albumin, and also uncover any possible cross talk between such pathways in the process.

Ellen Nollen et al. (2003) International Worm Meeting "Genome-wide RNAi screen for genes involved in polyglutamine aggregate formation"

Ronald H Plasterk, Richard I Morimoto, Gijs van Haaften, Susana Garcia, Ellen Nollen

[International Worm Meeting, 2003]

Accumulation of polyglutamine containing proteins into intracellular aggregates is associated with various CAG trinucleotide expansion disorders, including neurodegenerative diseases such as Huntingtons disease and spinocerebellar ataxias. The aggregation properties of polyglutamine proteins are directly related to the length of the polyglutamine stretch. With polyglutamine stretches above a threshold of approximately 30 glutamine residues, the aggregation rate increases with increasing numbers of glutamine residues. The length-dependent kinetics of aggregation recapitulates the length-dependent increase in cellular toxicity and age of onset of disease. Expression of polyglutamine stretches of 0, 24, 33, 35, 40, 44, and 82 glutamine residues as YFP-fusion proteins under control of the muscle specific unc-54 promoter in C. elegans reconstitutes the length and age dependence of aggregation. 1 Whereas worms expressing YFP-fusions with polyglutamine stretches up to 24 (Q24) show a diffuse YFP staining in all muscle cells, Q82 animals show a punctate staining in most of the cells. Interestingly, all Q lengths show variability in aggregation within individual animals, depending on the cell and the age of the worm, which is influenced by the genetic background of the worms. For example, aggregation of Q82-YFP is greatly delayed in the aging mutant age-11. This heterogeneity of aggregation suggests that genes exist that influence the formation of polyglutamine aggregates. We are using a genome-wide RNAi screen to identify genes involved in polyglutamine aggregation. In a candidate gene approach with RNAi against genes encoding molecular chaperones and molecules involved in proteins degradation, we have already identified genes that may play a role in aggregate formation. 1Morley JF, Brignull HR, Weyers JJ, Morimoto RI. Proc Natl Acad Sci U S A 2002 99(16):10417-22

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.