Preferential cleavage sites for Sau3A restriction endonuclease in human ribosomal DNA

Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in r epresentativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvo lodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The h ypersensitive cleavage sites were found to be located in the ribosomal inte rgenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb up stream of the rRNA transcription start point. This finding is based on sequ encing mapping of the rDNA insert ends in randomly selected cosmid clones o f human chromosome 13 and on the data of digestion kinetics of cloned and n oncloned human genomic rDNA with Sau3A and MboI. The results show that a me thylation status and superhelicity state of the rIGS have no effect on clea vage site sensitivity. It is interesting that all primary cleavage sites ar e adjacent to or entering into Alu or psi cde 27 retroposons of the rIGS su ggesting a possible role of neighboring sequences in nuclease accessibility . The results explain nonequal representation of rDNA sequences in the huma n genomic DNA library used for this study. (C) 2000 Academic Press.