temperature-compensated ultradian clock in Caenorhabditis elegans

Substantial progress has been made in understanding the genetic basis of temperature-compensated circadian clocks. Ultradian rhythms, with a period shorter than 24 h, are at least as widespread as circadian rhythms. We have initiated genetic analysis of defecation behavior, which is controlled by an ultradian clock in Caenorhabditis elegans. The defecation motor program is activated every 45 sec, and this rhythm is temperature compensated. We describe mutations in 12 genes that either shorten or lengthen the cycle period. We find that most of these mutations also disrupt temperature compensation, suggesting that this process is an integral part of the clock These genes open the way for molecular genetic dissection of this ultradian clock Rhythmic activities are found universally from single-celled organisms to multicellular eukaryotes (1-6). Genetic and molecular analyses of one type of biological rhythm, the circadian rhythm, have been successfully carried out in several organisms, including cyanobacterium (7), Drosophila (2-4), Neurospora (2-4), mouse (8), and Arabidopsis (9, 10). Circadian clocks have well-defined characteristics (1-4); the 24-h rhythm persists without environmental cues; the rhythm can be entrained by environmental cues; the oscillation phase can be reset by external stimuli, such as light or temperature; and the rhythm is nearly constant at different temperatures (temperature compensation). Ultradian rhythms, with a period of less than 24 h, are widely observed (5, 6). Some ultradian rhythms, including those of heart pacemaker cells and central pattern generator neurons, have been studied electrophysiologically (5, 11). The courtship song of Drosophila varies rhythmically with a period of 50 to 65 sec. This rhythm is temperature-compensated and has been reported to be affected by per circadian clock mutations (12). To systematically investigate the genetic basis of an ultradian clock, we studied defecation behavior, which, in the nematode Caenorhabditis elegans, is controlled by a temperature-compensated ultradian clock (13). With abundant food, a defecation motor program is activated every 45 sec, the rhythm persists in the absence of motor program activation, the oscillation phase can be reset by sensory stimuli, and the rhythm is nearly constant at temperatures from 19°C to 30°C (13). Here, we report that the defecation clock can be genetically dissected. We found that mutations in 12 genes cause abnormal defecation cycle periodicity (Dec phenotype). These mutations fall into two major groups: short Dec (Dec-s) and long Dec (Dec-f). Since most Dec mutations affect the cycle period differently at different temperatures, it is likely that these mutations affect temperature compensation rather than causing thermolabile gene products. This nematode defecation system is an excellent genetic model for studying an ultradian clock and temperature compensation. MATERIALS AND METHODS Genetics. All strains were derivatives of the Bristol C. elegans strain N2, and maintenance and handling of these strains were described previously (14). This paper follows the standard C. elegans nomenclature (15). To isolate dec mutants, young adult N2 hermaphrodites were treated with 50 mM ethyl methanesulfonate (14). Five mutagenized animals were picked and placed on a fresh 60-mm plate seeded with Escherichia coli. After these parents laid 20-30 eggs each, the parents were removed from the plate. When the F1 progeny became young adults, theywere transferred to fresh 60-mm plates seeded with E. coli (five animals per plate). After these five animals laid 20-30 eggs each, the F1 animals were removed. From each plate, five F2 progeny at the young adult stage were briefly observed by using a dissecting microscope. If an individual showed a potentially abnormal defecation cycle period, either shorter than 36 sec or longer than 72 sec, it was picked and placed on a fresh plate as a mutant candidate. Progeny from such candidates were examined more extensively. A total of 7000 F2 progeny of 7000 F1 animals were screened, a number equivalent to approximately 3000 mutagenized haploid genomes (16). Some mapping results for newly isolated genes are summarized below. A complete set of map data has been submitted to the Caenorhabditis Genetic Center and to the C. elegans data base (ACeDB). To avoid marker effects, recombinants with markers were crossed with Dec males and cycles were scored in cross progeny. All Dec-( mutants grew slowly. This slow-growing phenotype was used for mapping and in each case was confirmed to be linked to the Dec-( mutation. dec-2 maps at the extreme right of chromosome III, to the right of unc-25. A total of 163 slow-growing progeny were picked from dpy-18(e364) unc25(e156)/dec-2: 126/163 segregated no Dpy and no Unc, 4/163 segregated Dpy Unc, and 33/163 segregated Dpy nonUnc. dec-4 maps between unc-5 and dpy-20 on chromosome IV. A total of 89 slow-growing progeny were picked from unc-S(e53) dpy-20(e1282)/dec-4: 79/89 segregated no Unc and no Dpy, 3/89 segregated Unc non-Dpy, and 7/89 segregated Dpy non-Unc. dec-7 maps between unc-32 and unc-47 on chromosome III: from unc-32(e189) dpy-18(e364)/ dec-7, 2/8 Unc non-Dpy recombinants segregated Dec-s and 4/5 Dpy non-Unc animals segregated Dec-s; from sma2(e502) unc-47(e307)/dec-7, 2/7 Unc non-Sma recombinants segregated Dec-s and 2/3 Sma non-Unc animals segregated Dec-s. dec-9 maps between daf-14 and dpy-20 on chromosome -IV: from unc-24(e138) daf-14(m77) dpy20(e1362)/dec-9, 8/8 Unc non-Daf non-Dpy recombinants segregated Dec-s and 1/3 Unc Daf-c non-Dpy recombinants segregated Dec-s. dec-10 maps between unc-115 and egl-15 on chromosome X: from unc-115(e2225) egl-15(n484)/dec10, 1/2 Unc non-Egl recombinants segregated Dec-s and 3/4 Egl non-Unc recombinants segregated Dec-s. dec-li maps Abbreviations: Dec, abnormal defecation cycle periodicity; Dec-s, short Dec; Dec-(, long Dec; pBoc, posterior body-wall muscle contraction; aBoc, anterior body-wall muscle contraction; Exp, expulsion. *To whom reprint requests should be addressed. 10317 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10318 Genetics: Iwasaki et al.