The DNA fragments produced by AluI and BstNI digestion of fixed mouse chromosomes.
AluI and BstNI restriction endonucleases were used to study cytological and biochemical effects on centromere DNA in fixed mouse chromosomes. These enzymes were employed, as it is known that AluI is incapable of attacking major satellite DNA, contrary to BstNI that is known to cut this DNA fraction into monomers of 234 bp. After digestion in situ, electrophoretic analysis was carried out to characterize the DNA purified (1) from the material remaining on the chromosomes and (2) from the material solubilized from chromosomes. The DNA was then transferred to a nylon filter and 32P-labelled major satellite DNA was used as a probe for hybridization experiments. Other preparations were simply stained with Giemsa after digestion in situ with AluI and BstNI. Our results show that although restriction endonuclease cleavage primarily depends on DNA base sequence, this factor is not always sufficient to explain nuclease-induced cytological effects. In fact, the structural organization of peculiar regions such as the centromeres of mouse chromosomes might affect cleavage efficiency when restriction enzyme digestion is performed in situ.
Mezzanotte
R
R
Istituto di Biologia Generale, Facoltà di Medicina, Università di Cagliari, Italy.
Rossino
R
R
Nieddu
M
M
Lopez-Fernandez
C
C
Gosalvez
J
J
D000818:Animals; D002460:Cell Line; D002503:Centromere / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004276:DNA, Satellite / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D051379:Mice; D009693:Nucleic Acid Hybridization
D004276:DNA, Satellite; D004247:DNA; C024669:endodeoxyribonuclease AluI; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific