Maize DNA extracted from W64A was digested with HaeIII, Sau3AI or TaqI restriction enzymes and fractionated by acrylamide gel electrophoresis. Five fragments that light up in ethidium bromide staining were separated (H1 and H2 from HaeIII digest, S1 from Sau3AI digest, T2 and T3 from TaqI digest) and cloned in E. coli by ligation to pUC8 plasmid. The fragments do not cross-hybridize to each other and have a length between 150 and 220bp.
Southern blot hybridizations of nick translated fragments to total digested DNA from maize lines differing in heterochromatin content were carried out (W64A, A69Y, Black Mexican without or with seven B chromosomes). The cloned fragments appear to have a lower reiteration frequency in Black Mexican than in the other lines, suggesting a relation between these sequences and the knob heterochromatin, the knobs being absent in Black Mexican lines.
The chromosomal localization of these repeated sequences was detected by in situ hybridization on meiotic spreads of maize lines with (W64A, KYS) and without (Black Mexican with two or three B's) knob heterochromatin. The S1 fragment hybridization sites were found in Black Mexican only on the B chromosomes, but spread on seven or more A chromosomes in W64A and KYS lines, with preferential hybridization to knob heterochromatin. In all lines investigated some of the A chromosomes have hybridization sites essentially in the centromeric region for H1 and T2 probes while B chromosomes have none. The T3 fragment hybridization induces groups of spots on the B chromosomes and on few A chromosomes with different number of sites among the lines investigated. Finally the H2 fragment strongly and preferentially hybridizes to the NOR region of chr. 6 and only to the centromeric region of B chromosomes. In summary the five repeated sequences have different clustering and unique or multiple hybridization sites on both A and B chromosomes.
A. Viotti, N. Pogna, E. Privitera and E. Sala
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