Chromosome in Caenorhabditis elegans

Twelve new X chromosome duplications were identified and characterized. Eight are translocated to autosomal sites near four different telomeres, and four are free. Ten include unc-l(+), which in wild type is near the left end of the X chromosome, and two of these, mnDp72(X;ZV) and m n D p 7 3 ( X f ) , extend rightward past dpy-3. Both mnDp72 and mnDp73 recombined with the one X chromosome in males in the unc-1-dpy-3 interval at a frequency 15to 30-fold higher than was observed for X-X recombination in hermaphrodites in the same interval. Recombinant duplications and recombinant X chromosomes were both recovered. Recombination with the X chromosome in the unc-1-dpy-3 interval was also detected for five other unc-l(+) duplications, even though their right breakpoints lie within the interval. In hermaphrodites, mnDp72 and mnDp73 promoted meiotic X nondisjunction and recombined with an X chromosome in the unc-1-dpy-3 interval at frequencies comparable to that found for X-X recombination; mnDp72(X;ZV) also promoted trisomy for chromosome ZV. A mutation in him-8 ZV was identified that severely reduced recombination between the two X chromosomes in hermaphrodites and between mnDp73 and the X chromosome in males. Recombination between the X chromosome and duplications of either the right end of the X or a region near but not including the left end was rare. We suggest that the X chromosome has one or more elements near its left end that promote meiotic chromosome pairing. N UMEROUS translocated and free duplications of chromosomal segments have been generated and characterized in the nematode Caenorhabditis eleguns (HERMAN, ALBERTSON and BRENNER 1976; HERMAN, MADL and KARI 1979; HODGKIN 1980; HERMAN, KARI and HARTMAN 1982; ROSE, BAILLIE and CURRAN 1984; ANDERSON and BRENNER 1984; ROSENBLUTH, CUDDEFORD and BAILLIE 1985; DELONG, CASSON and MEYER 1987; ROGALSKI and RIDDLE 1988). Such duplications have been used to vary the dosage of genes (JOHNSON et al. 198 1 ; GREENWALD, STERNBERG and HORVITZ 1983; MEYER and CASSON 1986; DONAHUE, QUARANTILLO and WOOD 1987), to balance recessive lethal and sterile mutations (MENEELY and HERMAN 1979, 1981; HOWELL et al. 1987), to facilitate manipulation of X-linked markers (HERMAN, ALBERTSON and BRENNER 1976), to vary the X chromosome-to-autosome ratio (MADL and HERMAN 1979; MENEELY and WOOD 1984, 1987; WOOD et al. 1985; MENEELY and NORDSTROM 1988), to mark particular chromosomes cytologically (ALBERTSON 1984), and to generate genetic mosaics (for review, see HERMAN 1989). Some of the X chromosome duplications that have been characterized are translocated to autosomes. The other X duplications and all autosomal duplications that have been studied are free chromosome fragments, as shown by genetic and cytological criteria. The relatively high frequency at which free duGenetics 121: 723-737 (April, 1989) plications have been recovered is probably at least in part attributable to the holokinetic (also referred to as diffuse centromeric) nature of C. elegans chromosomes (ALBERTSON and THOMSON 1982). Nearly all duplications that have been studied previously, whether free or translocated, appear to recombine very infrequently with the homologous segments of the normal chromosomes. Those rare duplications that have been shown to recombine with their normal homologues are large; sDpl(Z;f) , which recombines with chromosome I over the dpy-5-unc-54 interval at moderate frequency, includes more than half of the genes on chromosome Z (ROSE, BAILLIE and CURRAN 1984), and m D p l , which recombines over the unc-17dpy-I3 interval of chromosome ZV at a frequency of 20-fold less than that found for chromosome-chromosome recombination, includes approximately half the genes on chromosome ZV (ROGALSKI and RIDDLE 1988). Examples of smaller duplications, for which recombination has been shown to be very rare, are duplications of the right end of the X chromosome. The rare apparently recombinant duplications of that region that were identified were at least in some cases, perhaps most, the result of mutation rather than recombination (HERMAN 1984). We do not understand why recombination between a small duplication and its homologous region of chromosome is generally rare. From a practical point of view, the low recombination frequency is a conven724 R. K. Herman and C;. I(. Kari ient property for most genetic applications of duplications. On the other hand, it is conceivable that a better understanding of the factors affecting duplication-chromosome recombination could help lead to a method for promoting gene disruption by homologous recombination of injected DNA fragments-a powerful tool in yeast genetics (ROTHSTEIN 1983) not yet available in C. eleguns although methods for promoting integrative transformation have been worked out (FIRE 1985). In this paper we show that certain small duplications, both translocated and free, of the left end. of the X chromosome recombine at surprisingly high frequencies with the homologous region of the normal X chromosome in both males (which carry a single X chromosome in addition to the duplication) and hermaphrodites (which have two X chromosomes). This is not a general property of X chromosome duplications because the frequencies of recombination between other small X duplications and the X chromosome appear to be very low in both males and hermaphrodites. We suggest that homologous pairing of the X chromosome of C. eleguns at meiosis is promoted by one or more sites near the left end of the X chromosome. MATERIALS AND METHODS Strains, genes and general procedures: C . elegans var. Bristol strain N 2 was the wild-type parent for all strains used in this work. Media, culture and mating techniques were as described by BRENNER (1974) and HERMAN (1978). The procedure used for staining chromosomes with DAPI (diamidinophenolindole) was identical to the previously-described procedure for staining with Hoechst 33258 (HERMAN, MADL and KARI 1979) except for the exchange of dyes. General gene names of genes used were: che, chemotaxis defective; dpy , dumpy: egl, egg-laying defective: j l u , abnormal gut autofluorescence; him, high incidence male self progeny; lin, cell lineage defective; Lon, long; mab, male abnormal; osm, defective in avoidance of high osmolarity; sup, suppressor: unc, uncoordinated. Names of genes and alleles used were: linkage group (LC) I : egl-30(n686), dpy-5(ehl), unc-54(e190). LGZIl: d p y l ( e l ) , unc-Y?(e1500n224) = unc-Y3(0) in the text, unc-Y3(e1500), unc-?6(e251). LGZV: him-8(e1489 or mn253), dpy-4(e1166). LGV: unc-60(e667), dpy-1 l(e224). IGX: mab-7(e1599). che-2(e1033), unc-l(e538), dpy-?(e27 or e182), lin-32(~282), unc-2(e55), osm-5(p81?), unc-20(e112), unc-78(e1217), lon-Z(e678), dpy-8(e130), jlu-2(e1003), unch(e78), dpy-7(e88), unc-Y(elOl), unc-84(e1410), unc-?(e151), unc-7(e5), osm-l(p808), sup-l0(n 183). A genetic map showing the relative positions of these genes is given as Figure 1 . For ;t more complete map and references to origins o f mutations, see WOOD (1 988). The position of sup-10 to the right o f osm-1 (A. VILLENEUVE and B. MEYER, personal cornmunication) is a correction on our earlier placement (MENEELY and HERMAN 198 1). The position of che-2 about 0.4 map uni t left of unc-1 is based on the following data: three of 340 Unc-l progeny of che-2 unc-lldpy-3 hermaphrodites were non-Che [as determined by staining with fluorescein isothiocyanate (FITC): see below], and none of the three segregated Dpy (Unc) animals. The position of mab-7 slightly left of unc-1 (and not resolved from the position of che-2) is based on the following data: among 424 male progeny generated by a cross between mab-7 dpy-3/ unc-1 hermaphrodites and N 2 males, one was Dpy non-Unc non-Mab and one was Mab Unc non-Dpy. The position of flu-2 (BABU 1974) between dpy-8 and unc-6 is based on the following data: among the progeny of dpy-8 unc-h/’u-2 hermaphrodites, 26 of 50 Dpy non-Unc and 16 of 50 Unc non-Dpy animals were Flu. The derivation of the duplication mnDp3 was described previously (HERMAN, MADL and KARI 1979). Genetic nomenclature follows the guidelines described by HoRvrrz et al. (1979), with the following addition: the genotype of a duplication is sometimes stated explicitly in brackets along with the duplication name, as, for example, mnDp62(X,f)[unc-?(+) osm-l(+) suplO(nl83)l. The che-2, osm-1 and osm-5 mutations abolish the uptake of FITC by amphid and phasmid sensory neurons (HEDGECOCK et al. 1985; PERKINS et al. 1986), and i t was this trait that we scored for these genes. The FITC staining protocol of HEDGECOCK et al. ( 1 985) was used. Some of our counts of Che-2 animals may be underestimates, by as much as about 20-30%, because of the suicidal tendency of these animals, particularly the males (HODGKIN 1983), to crawl off the agar plate. The mab-7 and lin-32 markers were scored, in males, by looking at the rays o f the copulatory bursa by Nomarski microscopy. I n mab-7 animals, the bllrsdl fan is reduced and the bursal rays are swollen (HODGKIN 1983); the lin-32 bursa is essentially devoid of rays (E. HEDGECOCK, personal communication). Induction, identification and characterization of uncI ( + ) duplications: The general strategies for generating and characterizingxduplications have been described (HERMAN, ALBERTSON and BRENNER 1976; HERMAN, MADI. and KARI 1979). N2 males were exposed to y-rays and mated with unc-1 hermaphrodites. The screen was greatly aided by the use of the recessive unc-1 allele e538 (PARK and HORVITZ 1986) in preference to the previous reference allele eY4, which is semidominant to unc-I(+). ?-Rays were supplied by “’Ck in a Shepherd irradiator (model 143-45). Doses of 4200 roentgens (r) were used at a dose rate of 530 r/min. Progeny were screened for exceptional wild-type males, which were mated with unc-1 hermaphrodites. When roughly half the male progeny of the latter cross were wild type, it w a s concluded that they carried a copy of unc-l(+) that was not X-linked. The frequency of recovery of uncI ( + ) duplications was about per male progeny. We learned from our first recovered unc-l(+) duplication, mnDp63, that many of the wild-type hermaphrodite progeny of a cross of unc-l/0:mnDp63 males and unc-I hermaphrodites did not carry mnDp63 but carried a recombinant uncI ( + ) X chromosome (see below). Therefore, i n order to guard against the possibility of losing duplication stocks, we maintained each duplication by continual backcrossing of wild-type males to unc-1 hermaphrodites. Each duplication was outcrossed dozens of times during the course of these experiments. T o test whether or not a given duplication carried the Xlinked gene g, wild-type males of genotype unc-l/O;Dp were crossed with g hermaphrodites. If roughly half (as opposed to virtually none) of the male progeny were non-G, it was concluded that the duplication carried g(+). The possible autosomal map location of each duplication was sought i n the following way: unc-I/O:Dp males were mated with m;unc-1 hermaphrodites, where m is an autosomal marker. Wild-type hermaphrodite progeny were picked. A potential difficulty at this stage was in identifying those wild-type hermaphrodites that carried the duplication (genotype m/I)p;unc-l/unc-I or m/+:Dp/+:unc-l/unc-1) rather than a recombinant X chromoson~e (genotype m/+;unc-l/ +). Different criteria for the presence o f different duplications were used. FOImnnp63, mnDp70 and mnDp72, the Dp-X Recombination in C. elegans 725