Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody.
The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A3 R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.
Ueda
T
T
Laboratory of Developmental Biology, Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan.
Kato
Y
Y
Irie
S
S
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology; D002460:Cell Line; D002725:Chloroform; D002871:Chromosome Banding; D002875:Chromosomes / Q000032:analysis*; D004247:DNA / Q000032:analysis*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D003670:Deer / Q000235:genetics*; D000431:Ethanol; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D008297:Male; D009690:Nucleic Acid Conformation
D000085:Acetates; D000974:Antibodies, Antinuclear; C033474:Carnoy's solution; D005404:Fixatives; D000431:Ethanol; D002725:Chloroform; D004247:DNA; D004264:DNA Topoisomerases, Type I; D019342:Acetic Acid