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In o current fine-rtwcture onolyris of the methionine-1 locus, a method of obtaining me-l with the desired right-hand ond left-hand outside markers has beendeveloped. This involves the use of the temperolure-sensitive colonial mutant cot. The method can be used with ony pair of markers proximal or disxto it. Using the cross me-l x hist-5,e os on exomple, the method is (IS follows: Ascorpores ore harvested with o loop of sterile dir-tilled water, ruspendedin 1-f sterile distilled water, heat-shocked for 30 minutes ot 60°C, and 0.5 ml of this suspension per plate is spread on several hirtidine-supplemented plater. A firmer medium (336 ogor) focilitotes isolation. These plates ore in-cuboted ot 32'C for eight to ten hours. Since the order is me-l-hist-5-cot, most of the orcosporer that grow will be the hist-5, cot poren+ols and one-holf the single crossovers in ra Z.Theregrozg orcorpora ore ignored, ond IOOof the germin-oted but non-growing oscorporer ore isolated to methionine plus hirtidine-supplemented tubes. These isolates ore incubated ot 32'C for 48 hours. The co++ isolates (those with wild-type morphology) ore discorded, and the cot-isolates ore allowed to grow up at 2YC. The latter ixter ore then tested on methionine-supplemented liquid medium, ond approximately 15% of them will show no growth, indicating that they ore the desired me-l, hirt-5, cot recombinonts.-If one wishes to obtain me-l with o porticulor left-hand maker, soy pyridoxine-I, the procedure is reversed. The orcorpores ore plated on methionine medivm. germinated but non-growing ascosporer ore isolated to methionine plus pyridoxine-rupplemented tuber, and the cot-isolates ore discorded. motely 12% of?&& remaining cot+ In this instance, upon testing on methionine-supplemented liTid medium, approxi-wlotes will show no growth ond be the desired e, me-l recombinontr.

A rapid method for obtaining linkage group IV double mutant stocks