Visualization of sister-chromatid exchanges in maize mitotic chromosomes utilizing 5-bromodeoxyuridine
We have recently applied the fluorescence plus Giemsa technique to maize roottip somatic chromosomes to visualize sister-chromatid exchanges (SCE's). Several different lines of corn have been used successfully with the protocol given below.
Kernels were germinated in the dark at 270 C. Primary roots about 2-3 cm long still attached to the kernel were immersed in an aqueous solution containing 300 然 5-bromodeoxyuridine, 0.1 然 5-fluorodeoxyuridine, and 5 然 uridine for one cell cycle (8.8 hr). They were then transferred to an aqueous solution containing 300 然 thymidine and 5 然 uridine for a second cell cycle. After the above treatment, roots were excised and treated with a 0.002 M 8-hydroxyquinoline solution for 3 hr, and then fixed in an ethanol-acetic acid (3:1 v/v) solution. Root-tips were stored in the fixative at -220 C. All treatments were carried out in the dark or under a safelight equipped with a Kodak OC filter at 270 C. After fixation, the roots were washed in a citric acid buffer (0.01 M, pH 4.7) and incubated for 60 min at 270 C with 0.5% pectinase (Sigma Chemical Co.) dissolved in the same buffer. Meristematic regions of root-tips were placed on a glass slide and macerated in 45% acetic acid. The coverslips were removed using dry ice and the preparations were passed through a 100, 95, 85, 70, 50, and 30% ethanol (v/v) series to distilled water. The slides were incubated for 60 min at 270 C in a RNase solution made up of 10 ml of 0.5xSSC (sodium saline citrate) containing 1 mg ribonuclease A type 1-A from bovine pancreas (Sigma Chemical Co.). The slides were then rinsed with 0.5xSSC and treated with a Hoechst 33258 solution in 0.5xSSC for 25 minutes at the same temperature. The Hoechst 33258 solution was prepared by dissolving 1 mg of the fluorochrome in 1 ml of absolute ethanol and 0.1 ml of this solution was added to 200 ml of O.5xSSC. A drop of 0.5 SSC was placed on the slide, a cover glass was placed over the cells, and the cover glass was sealed with rubber cement. The slides were then placed 10 cm from a fluorescent sun lamp (Westinghouse FS 20) for 13 hr. After exposure, slides were incubated at 550 C in 0.5xSSC for 1.5 hr, stained with 3% Giemsa stain in phosphate buffer at pH 6.8 for 8 minutes, and washed in the same buffer, air dried, and mounted in Permount (Fisher).
After this treatment, the unifiliary substituted chromatid can clearly be distinguished from the unsubstituted chromatid of each chromosome and sister-chromatid exchanges are easily identified.
Black Mexican sweet corn without B chromosomes has been examined. Of 390 chromosomes, 59 had one, 13 had two, and one had three sister chromatid exchanges. Thus, 88 SCE's were present in 390 chromosomes scored giving a frequency of 0.22 SCE's per chromosome.
Tau-San Chou and David Weber
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