Medizin - Open Access LMU - Teil 05/22 Archiv
250 Episoden
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Cells of synchonized and of asynchronously growing cultures of a V79 Chinese hamster line were microirradiated with a low poweer laser-UV-microbeam of wavelength 257 nm. Ultraviolet light was either focused onto a small part of the nucleus (mode I) or distributed over the whole nucleus (mode II). Following microirradiation, the cells were incubated for 7–20 h with caffeine (1–2 mM) until chromosome preparation was performed. After both modes of microirradation, shattering of the entire chromosome complement (generalized chromosome shattering, GCS) was observed. It is suggested that the probability by which GCS is induced depends on the total number lesions rather than on their distribution in the chromatin. The results are consistent with the prediction of a “factor depletion model” wich assumes that in a given cell, GCS takes place both in irradiated and non-irradiated chromosomes of the total number of daughter strand-repair sites supasses a threshold value.
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A laser microbeam apparatus, based on an excimer laser pumped dye laser is used to microdissect human chromosomes and to isolate a single chromosome slice.
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The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.
