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 representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, 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 hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study.
Kupriyanova
N S
NS
Institute of Gene Biology, Russian Academy of Sciences, Vavilov Str. 34/5, Moscow, 117334, Russia. dna@biogen.msk.su
Kirilenko
P M
PM
Netchvolodov
K K
KK
Ryskov
A P
AP
D002882:Chromosomes, Human, Pair 13; D003360:Cosmids / Q000378:metabolism; D004251:DNA Transposable Elements; D004275:DNA, Ribosomal / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008745:Methylation; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004275:DNA, Ribosomal; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases