Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy.
The recent application of DNA cloning and non-radioactive in situ hybridization techniques has strengthened the hypothesis of an ordered chromatin structure in interphase nuclei. The arrangement of specific chromosomal regions is not random and is strongly suspected to vary with functional activity. The combination of in situ hybridization and immunocytochemistry, allowing simultaneous detection of nucleic acid sequences and specific antigens in the same nucleus, has already made significant contributions to the study of gene expression, to simultaneous karyotyping and phenotyping of tumor cells, and to in situ analysis of viral infections. This report emphasizes the considerable interest of such combined techniques for functional in situ mapping of the genome at the individual cell level. We propose a method that combines fluorescence immunocytochemical detection of nucleolar proteins and fluorescence in situ hybridization of centromeric and telomeric probes specific for chromosome 1 in two cultured human cell lines. The preparative constraints for a broad application of this procedure are defined so that the cell preparations can be further analyzed by fluorescence microscopic imaging techniques and confocal laser scan microscopy. The two selected sequences of the human chromosome 1 can be localized in the nucleus with respect to nucleolar proteins in a one-step fluorescence microscopic observation.
Leger
I
I
Equipe de Reconnaissance des Formes et de Microscopie Quantitative, CERMO, Université Joseph Fourier, Grenoble, France.
Robert-Nicoud
M
M
Brugal
G
G
D002466:Cell Nucleolus / Q000737:chemistry; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1 / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence / Q000379:methods*; D009687:Nuclear Proteins / Q000032:analysis*; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D004247:DNA