A new, improved FPG technique for detecting sister-chromatid exchanges in mitotic chromosomes

We have reported a fluorescence-plus-Giemsa (FPG) technique for visualizing sister-chromatid exchanges (SCE's) in maize mitotic chromosomes utilizing 5-bromodeoxyuridine (MNL 54:88-89, 1980). Recently, certain modifications of the maize FPG technique have been developed. The modifications involved pectinase concentration and treatment time, the concentration of Hoechst 33258 stain, and the temperature during the post-stain treatment.

In the previously described procedures, the pectinase concentration was 0.5% and the treatment time was 1 hr. Under this treatment condition, root tips were still relatively hard. After maceration by a glass rod, many metaphase cells were broken. The broken cells may be the cause of chromosome loss during the later slide preparation procedures. Softening the root tips by pectinase treatment may increase the percentage of intact cells after slide preparation. For this reason, various enzyme concentrations and treatment times were tested. When the pectinase concentration was 1.5% and the treatment time was 2 hrs., the root tips were soft enough to be smashed with a dissecting needle. Then, cells were gently macerated with a glass rod and flattened by placing a cover glass on the suspension and pressing on it. After these treatments, many intact metaphase cells were observed. Intact or slightly damaged cells had a better chance of staying on a slide than chromosomes from ruptured cells during the treatments required to demonstrate sister chromatid differentiation. Goto et al. (Chromosoma 66:351-359, 1978) suggested a practical concentration of 10-5 M of Hoechst 33258 to detect SCEs in rat bone marrow cells. This is ten times higher than the concentration we reported previously To make the 10-5 M Hoechst 33258 solution, 1 mg of Hoechst 33258 was dissolved in 1 ml of 100% ethanol, and 1 ml of this solution was added to 200 ml of 0.5 x SSC.

Another factor that affects the success of the sister-chromatid differentiation is the temperature during the UV exposure. In our previous report, the experiments were performed at room temperature. However, the temperature in the laboratory fluctuated during the day and between seasons. In our modified procedure, slides were placed on a slide warmer and this kept the temperature at 45 C during UV exposure.

The rest of the slide preparation procedures were the same as previously described. A far higher percentage (20-40 times as many) of the cells treated with this improved protocol, showed a better sister-chromatid differentiation than with the protocol initially developed.

Tau-San Chou and David Weber
 
 


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