Methylation-sensitive restriction endonuclease digestion patterns revealed in Vicia faba L. chromosomes by in situ nick-translation.
Restriction endonucleases sensitive to cytosine methylation (HpaII, MspI and HhaI) and 5-azacitidine were used to study the localization of target sequences in Vicia faba metaphase chromosomes by in situ digestion and radioactive or non-radioactive nick-translation. In control experiments, neither isolated DNA nor chromosomes in situ were digested by HpaII and MspI. Pretreatment with demethylating agent, 5-azacitidine resulted both in increased effectiveness of in situ digestion and nick-translation. In 5-azacitidine-treated material, negative bands in M chromosomes appeared. HhaI cleaved isolated DNA, digested it in situ and gave positive signals as a result of nick-translation procedure in metaphase chromosomes. In S chromosomes containing heterochromatin without target sequences for HpaII and MspI, negative bands were shown after nick-translation. Such heterochromatin contains FokI sequences and in situ nick-translation driven by that restriction enzyme resulted in positive bands.
Olszewska
M J
MJ
Department of Plant Cytology and Cytochemistry, University of Lodź, Poland.
Gernand
D
D
Sakowicz
T
T
D001374:Azacitidine / Q000494:pharmacology; D002871:Chromosome Banding / Q000379:methods; D019175:DNA Methylation; D018744:DNA, Plant / Q000378:metabolism*; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015183:Restriction Mapping / Q000379:methods*
D018744:DNA, Plant; D004791:Enzyme Inhibitors; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; D001374:Azacitidine