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A Genetic Mapping System in Caenorhabditis Elegans Based On z A Genetic Mapping System in Caenorhabditis elegansBased on Polymorphic Sequence-Tagged Sites Benjamin D. Williams,* Bertold Schrank,* Chau Huynh,* Ratna Shownkeent and Robert H. Waterston* *Department of Genetics, Washington University School ofMedicine, St. Louis, Missouri631 IO, and ?Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom Manuscript received December 26, 1991 Accepted for publication March 9, 1992 ABSTRACT We devised an efficient genetic mapping system in the nematode Caenorhabditis elegans which is based upon the differences in number and location of the transposable element Tcl between the Bristol and Bergerac strains. Using the nearly completed physical map of theC. elegans genome, we selected 40 widely distributed sites which contain a Tcl element in the Bergerac strain, but not in the Bristol strain. For each site a polymerase chain reaction assay was designed that can distinguish between the Bergerac Tcl-containing site and the Bristol “empty” site. By combining appropriate assays in a single reaction, one can score multiple sites within single worms. This permits a mutation to be rapidly mapped,first to a linkage group and thento a chromosomal subregion, through analysis of only a small number of progeny from a single interstrain cross. EQUENCE-tagged sites (STSs), which havere- Previously, Tcls have been used in efforts to mo- S cently been proposed as common landmarks for lecularly clone specific genes, both by direct transpo- genomic physical mapping (OLSONet al. 1989), are son tagging(MOERMAN, BENIAN and WATERSTON short, unique genomic regions that areeasily detected 1986) andby genetic mappingof the Bristol/Bergerac by polymerase chain reaction(PCR) amplification Tcl polymorphisms nearest a gene to identify molec- (SAIKIet al. 1988). A primary virtue of STSs is that ular clones from the region (RUVKUN et al. 1989). they can be distributed among laboratoriesas a DNA Conversely, cloned genes have been placed on the sequence database, obviatingthe transfer and storage linkage map by using Tcl polymorphisms as genetic of biological materials. tags (FILES,CARR and HIRSH1983). Polymorphic genomic regions have also been con- To develop a genome-wide genetic mapping strat- verted into STSs by appropriately designed PCR as- egy, individual randomly cloned Bergerac Tcls were says. These arevaluable as easily scored genetic mark- positioned within the nearly completed C. elegans ge- ers andas points of correspondence between physical nomic physical map (COULSONet al. 1986,1988, and genetic maps. Variable numbers of repetitive 1991). PCR assays were then developed for a subset mono-, di- and trinucleotide motifs (LITT and LUTY of the Tcls, selected to give markers well distributed 1989; TAUTZ1989; WEBERand MAY 1989), referred throughout the genome. The assays yield a product to as microsatellites, and variable lengths of the poly- when the Tcl is present (Bergerac DNA) but give no deoxyadenylate tracts within individual Alu elements product when the Tcl is absent (Bristol DNA). The (ECONOMOUet al. 1990) are examples of polymor- resulting STS map has sufficient density to allow a phisms that have been rapidly scored through appro- new mutation to be mapped to a chromosomal subre- priatelydesigned PCRassays. Alarge number of gion through analysis of a limited number of progeny microsatellites in the mouse genome have been sys- from a single interstrain cross. Since the STS markers tematically cloned and converted into polymorphic do nothave an associated phenotype and can be STSs with the goal of producing a polymorphic STS scored directly by PCR with DNA released from single map (CORNALLet al. 1991). animals or embryos, they offer advantages beyond a Here we report the systematic development of a simple reduction in the number of crosses needed to polymorphic STS map in the nematodeCaenorhabditis map new mutations. These are demonstrated herein elegans. The polymorphisms exploited in this scheme mapping experiments with lethal and X-linked muta- are copies of the transposable element Tcl which are tions. present at dispersed loci in the high Tcl-copy number Bergerac strain (approximately 500 copies), but ab- MATERIALS AND METHODS sent from these same locations in the low Tcl-copy Strains,genes and mutations: All mutations used in number Bristol strain (30copies) (EMMONSet a2. 1983; mappingexperiments were generated in theBristol N2 LIAO,ROSENZWEIG and HIRSH 1983). background. Most have been described by BRENNER(1 974) Genetics 131: 609-624 uuly, 1992) 610 D. B. Williams et al. and by HODCKINet al. (1988); the genes and corresponding to a sequence within the Tcl element, and primers specific mutations are listed below for each chromosome. for each Tcl insertion site. Each of the latter is complemen- Chromosome I: dpy-5(e61), unc-Z9(e1072), unc-75(e950), tary to DNA sequences that flank the Tc 1 insertion site and unc-lOl(ml), unc-95(su33), lev-1 l(x1.2). will work incombination with primer 6 18 to amplify a DNA Chromosome 11: sqt-2(sc3), dpy-1 O(el28}, unc-4(e 1 ZO), unc- fragment of defined size from the Bergerac Tc1 -containing 52(st572) (B. WILLIAMSand R. WATERSTON,unpublished site, but not from the "empty" Bristol site. results), unc-52(e444). DNA for PCR assay wasprepared from individual animals Chromosome III unc-45(e286ts), dpy-l(el), dpy-l8(e364). using methods adapted from BARSTEAD,KLEIMAN and WA- Chromosome IV dpy-9(e12), deb-l(st385) (BARSTEADand TERSTON (1991). Singleanimals were picked up with a WATERSTON1991), unc-ZZ(s12). platinum wire and each placed in a 2.5-p1 drop of lysis buffer Chromosome V unc-60(e677), dpy-1 I(e224}, unc-Z3(e25), [60 cg/ml proteinase K in 10 mM Tris (pH 8.2), 50 mM KCl, dpy-Zi(e428), rol-9(sc148}. 2.5 mM MgC12, 0.45% Tween 20 and 0.05% gelatin) in the Chromosome X dpy-3(&7), unc-Z7(e155), unc-90(e1463), cap of a separate 0.5-ml tube suitable for PCR. The drops patfst558) (B. WILLIAMSand R. WATERSTON,unpublished were then moved to the bottom of the tubes by a brief results). microfuge spin, frozen (-70°, 15 min), and after the addi- Two Bergerac strains, RW7000 and DP13, were used to tion of a mineral oil overlay, heated (SO", 1 hr followed by generate the Bristol/Bergerac hybrid animals required for 95", 15 min). After cooling to 4", 22.5 pI of a "master mix" all polymorphic STS mapping experiments. DP13 is a deriv- were pipetted on top of the mineral oil overlay. The mix is ative of RW7000 obtained after maintenance for several formulated to bring the reaction volume to 25 pl with these years asan independent strain in another laboratory. There final conditions: 25 pmol of internal Tcl primer 618 and are only two differences between the strains with respect to 25 pmol each for the appropriateflanking sequence primers, the STS polymorphisms described here: (1) Tc 1 eP64 III is 10 mM Tris (pH 8.3), 50 mM KCI, 1.5 mM MgC12, 0.001% present in DP13 but absent from RW7000, and (2) Tcl (w/v) gelatin, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, stP128 V is present in RW7000, but absent from DP13 (see 0.2 mM dGTP and 0.6 unit Taq polymerase (Perkin Elmer RESULTS). Cetus, Norwalk, Connecticut). A brief microfuge spin was Techniques for culture and genetic analysis were as de- used to move the "master mix" through the mineral oil scribed by BRENNER(1974). All crosses were at 20" except overlay, and the reactions were rapidly heated (within 1 for those with unc-45(e286ts) homozygotes, which were set min) to 94" and cycled 30 times: 94" for 30 s, 58" for 1 up at 15"and then shifted to 20" after 2 days. min, and 72 O for 1 min. Failure to promptly bring the tubes Cloning, physical mapping and sequencingof Bergerac to 94" after the brief microfuge spin results in a significant Tcl insertion sites:Most of the Bergerac Tc1 s were isolated increase in background bands that can complicate interpre- from a library of RW7000 genomic DNA in vector X1059 tation of the data. Gel lanes were loaded with 10 pl of each by hybridization with Tcl sequences (I. MORI,D. MOERMAN reaction. and R. H. WATERSTON,unpublished results). DNA mini- Individual unhatched late-stage embryos were also used preps (HELMSet al. 1987) from positive clones were sub- for single PCR reactions. Eggs were picked up directly from jected to fingerprint analysis (COULSONet al. 1986) to place a culture plate using a 5-pl capillary tube which had been them in the C. elegans genomic physical map. Clones which pulled to a convenient inner diameter (50-100 pm) and could not be positioned by fingerprinting were radiolabeled filled with chitinase solution [20 mg/ml chitinase (Sigma C- by random priming (Prime-It kit, Strategene, La Jolla, Cal- 6137) in 50 mM NaCI, 70 mM KCI, 2.5 mM MgCh and 2.5 ifornia) and hybridized using standard procedures to a grid- mM CaCI2]. They were then immediately transferred to ded set of YAC clones that correspond to most of the C. worm lysis buffer, and processed as described above. The elegans physical map (COUUON et al. 1991). For clones same capillary tube was used for multiple transfers without mapping to advantageous positions, the sequence of DNA significant cross-contamination of DNA. flanking the Tcl was determined directly from miniprep Amplification products were separated using standard DNA using primer 61 8 (see Table l), which is complemen- nondenaturing 6% acrylamide minigels (Mighty Small 11, taryto Tcl sequence (ROSENZWEIG,LIAO and HIRSH Hoeffer Scientific Instruments, San Francisco, California). 1983a), by using a linear amplification sequencing method Mapping of new mutations: Since Bergerac males of the (MURRAY1989), as modified (CRAXTON1991).
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