METHYLATION STATUS OF RIBOSOMAL-RNA GENE CLUSTERS IN THE FLOW-SORTED HUMAN ACROCENTRIC CHROMOSOMES

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.