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Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system


, : Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system. Cytometry 13(5): 453-461

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were .apprx. 0.63 .+-. 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number. The relative increase in copy number indicated was similar to that reported in the literature.

US$19.90

PMID: 1321707

DOI: 10.1002/cyto.990130502


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