In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment.
Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.
Centre of Human Genetics and Genetic Counselling, University of Bremen, Federal Republic of Germany.
S
S
Bartnitzke
D
D
Eberhardt
J
J
Krooss
B
B
Rommel
J
J
Bullerdiek
D002871:Chromosome Banding; D002877:Chromosomes, Human / Q000187:drug effects* / Q000648:ultrastructure; D004247:DNA / Q000187:drug effects / Q000648:ultrastructure; D015257:DNA-Cytosine Methylases / Q000494:pharmacology*; D019286:Endopeptidase K; D006570:Heterochromatin / Q000187:drug effects; D006651:Histocytochemistry / Q000379:methods; D006801:Humans; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000494:pharmacology
D006570:Heterochromatin; D004247:DNA; C054926:DNA modification methylase AluI; D015257:DNA-Cytosine Methylases; D012697:Serine Endopeptidases; D019286:Endopeptidase K