(e.g.,Mather1936;ShermanandStack1995;Sy-Mapping the physical distribution of crossing overbenga1996).along chromosomes is an important step toward under-In general, the pattern of crossover events on chromo-standing the regulation of crossing over. Many physicalsomes can be summarized as follows:maps of recombination have been based on chiasmata,1. Ordinarily, each synapsed pair of homologous chro-with the most precise maps having been prepared formosomes (bivalent) has at least one crossover, re-grasshoppers and locusts, in which diplotene chiasmatagardless of the length of the bivalent (Mather1936).are unusually distinct (e.g.,Henderson1963;Fox1973;2. After the “obligate” crossover, the number of addi-ShawandKnowles1976;LaurieandJones1981).tional exchange events is proportional to chromo-More recently, electron microscopy has been used tosomal length;i.e., physically longer chromosomes arevisualize and map late RNs on SCs of several organismsmore likely to have more than one crossover (e.g.,(e.g.,Carpenter1975;Zickler1977;Carmi1978;HolmMather1936, 1937;Kabacket al.1992).andRasmussen1980, 1983;Glamann1986;Sherman3. A crossover in one region of a chromosome reducesandStack1995). Although the majority of RN mapshave been made using three-dimensional reconstruc-tions of serially sectioned nuclei, SC spreads are muchCorresponding author:Lorinda Anderson, Department of Biology,easier to produce and analyze than three-dimensionalColorado State University, Fort Collins, CO 80523.E-mail: [email protected]reconstructions (RahnandSolari1986;Stacket al.Genetics151:1569–1579 (April 1999)
1570L. K. Andersonet al.aqueous solution of 1% paraformaldehyde (pH 9.2) and1993;PigozziandSolari1997). However, late RNs are0.15% Triton X-100 that contained a cocktail of protease inhib-only rarely observed in SC spreads from mammals (e.g.,itors (final concentration 0.1mg/ml each for pepstatin A,Poormanet al.1981;Moenset al.1987), so this formchymostatin, antipain, and leupeptin, and 1mg/ml for aproti-of high-resolution physical crossover mapping has notnin) was dropped onto each slide. The supernatants wereremoved, and the pelleted cells in each microtube were resus-been practical for this group of animals.pended in 40ml of 0.1msucrose with the same proteaseAn alternative approach for mapping crossover sitesinhibitor cocktail to give a final total volume of 240ml. A 20-on mammalian chromosomes recently became possibleml aliquot of cell suspension was placed on each of 12 slides,using fluorescent immunological detection of MLH1and a cover was placed over the plastic dish so the slides would(MutL homolog) protein on SC spreads. MLH1 is anot dry. After 2 hr, the slides were gently rinsed in 0.4%Photoflo 200 and air dried. Unstained slides were examinedmismatch repair protein that is similar to the bacterialusing phase microscopy, and only slides on which the cellsMutL protein (Bronneret al.1994;Bakeret al.1996).