Macromolecular organization of human centromeric regions reveals high-frequency, polymorphic macro DNA repeats

To analyze the macromolecular organization of human centromeric regions, we used α-satellite, or alphoid, repetitive DNA sequences specific to the centromeres of human chromosomes 6 (D6Z1), X (XC), and Y (YC-2) and the technique of pulsed-field gel electrophoresis. Genomic DNA from 24 normal, unrelated individuals was digested and separated into fragments ranging from 23 kilobases (kb) to 2 megabases (Mb) in length. Digestion with 12 different restriction enzymes with 4- to 8-base-pair recognition sequences and hybridization with alphoid sequences revealed chromosome-specific hybridization patterns. Similarities in the organization of the centromeric regions of the three chromosomes included Not I, Sfi I, and Sal I fragments of > 2 Mb and Sau3A1 and Alu I fragments of < 150 kb. Each restriction enzyme with a 6-base-pair recognition sequence (Ava II, BamHI, HindIII, Hpa I, Pst I, Sal I, Sst I, and Xba I) detected polymorphic DNA fragments of 50 kb to 2 Mb. Forty percent or more of the individuals screened revealed a unique hybridization pattern with these enzymes and at least one of the three chromosome-specific alphoid probes. Five individuals differed from one another in hybridization pattern for each of the three enzymes HindIII, Hpa I, and Sst I and for each of the three centromeric probes. All 24 individuals could be distinguished on the basis of unique hybridization patterns with only two enzymes and one chromosome-specific alphoid probe. Family studies showed that these polymorphisms are inherited. The high frequency of these macro restriction fragment length polymorphisms illustrates the high degree of variability of the centromeric region among normal individuals and demonstrates its usefulness for DNA fingerprinting and pericentromeric mapping by linkage analysis.