# Eg mather 1936 sherman and stack 1995 sy mapping the

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( e.g. , Mather 1936; Sherman and Stack 1995; Sy- Mapping the physical distribution of crossing over benga 1996). along chromosomes is an important step toward under- In general, the pattern of crossover events on chromo- standing the regulation of crossing over. Many physical somes 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 for mosomes (bivalent) has at least one crossover, re- grasshoppers and locusts, in which diplotene chiasmata gardless of the length of the bivalent ( Mather 1936). are unusually distinct ( e.g. , Henderson 1963; Fox 1973; 2. After the “obligate” crossover, the number of addi- Shaw and Knowles 1976; Laurie and Jones 1981). tional exchange events is proportional to chromo- More recently, electron microscopy has been used to somal length; i.e. , physically longer chromosomes are visualize and map late RNs on SCs of several organisms more likely to have more than one crossover ( e.g. , ( e.g. , Carpenter 1975; Zickler 1977; Carmi 1978; Holm Mather 1936, 1937; Kaback et al. 1992). and Rasmussen 1980, 1983; Glamann 1986; Sherman 3. A crossover in one region of a chromosome reduces and Stack 1995). Although the majority of RN maps have been made using three-dimensional reconstruc- tions of serially sectioned nuclei, SC spreads are much Corresponding author: Lorinda Anderson, Department of Biology, easier to produce and analyze than three-dimensional Colorado State University, Fort Collins, CO 80523. E-mail: [email protected] reconstructions ( Rahn and Solari 1986; Stack et al. Genetics 151: 1569–1579 (April 1999)
1570 L. K. Anderson et al. aqueous solution of 1% paraformaldehyde (pH 9.2) and 1993; Pigozzi and Solari 1997). However, late RNs are 0.15% Triton X-100 that contained a cocktail of protease inhib- only rarely observed in SC spreads from mammals ( e.g. , itors (final concentration 0.1 m g/ml each for pepstatin A, Poorman et al. 1981; Moens et al. 1987), so this form chymostatin, antipain, and leupeptin, and 1 m g/ml for aproti- of high-resolution physical crossover mapping has not nin) was dropped onto each slide. The supernatants were removed, and the pelleted cells in each microtube were resus- been practical for this group of animals. pended in 40 m l of 0.1 m sucrose with the same protease An alternative approach for mapping crossover sites inhibitor cocktail to give a final total volume of 240 m l. A 20- on mammalian chromosomes recently became possible m l aliquot of cell suspension was placed on each of 12 slides, using fluorescent immunological detection of MLH1 and a cover was placed over the plastic dish so the slides would ( M u tL h omolog) protein on SC spreads. MLH1 is a not dry. After 2 hr, the slides were gently rinsed in 0.4% Photoflo 200 and air dried. Unstained slides were examined mismatch repair protein that is similar to the bacterial using phase microscopy, and only slides on which the cells MutL protein ( Bronner et al. 1994; Baker et al. 1996).