Directed Chromosome Loss by Laser Microirradiation

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Abstract
In this article I have presented data that indicate the feasibility of attaining the five objectives outlined in the introduction. It should be possible to assign genes to specific chromosome regions by (i) selective DNA deletion of a 0.25- to 0.5-µ.m segment of one or both homologous chromosomes, (ii) deletion of one or both entire homologous chromosomes, or (iii) combining cell fusion with selective deletion of whole chromosomes and then deletion of chromosome segments. By laser microirradiation it should be possible to determine which chromosomes and chromosome regions are essential for immediate cell survival by removing from individual cells whole chromosomes, and chromosome segments from each of the chromosomes in the karyotype, and then assessing the cloning efficiency of each cell. For example, we have already determined that removal of one large chromosome No. 1 from PTK2 cells does not prevent the cell from undergoing a subsequent mitosis. It should also be possible to generate new classes of mutants by damaging small selected areas of DNA with the laser beam and then cloning the irradiated cells—but this has yet to be demonstrated. This procedure might reveal recessive alleles on the nonirradiated homolog, or might result in the direct production of a genetic mutation. Irradiation of identical places on both homologous chromosomes could result in deletion of a genetic locus which ultimately might be detected as a deficiency in a metabolic pathway or some other cellular abnormality. Studies on chromosome stability and DNA constancy can be conducted with laser irradiated cells. For example, the karyotypic analysis of chromosome No. 1 suggests that a cellular mechanism exists to maintain the constancy of this chromosome in both the diploid and tetraploid cell lines. The same approach could be used with each of the chromosomes in the karyotype. Various cytochemical procedures could be used for making quantitative DNA studies of the cells, and chromosome and DNA analyses could be performed at varying times following laser microirradiation. It might also be possible to study the repair of chromosomal damage caused by laser irradiation. The cells could be examined by autoradiographic, cytochemical, and electron microscopy procedures at varying times after irradiation, and because the precise location, time, and nature of the mutational event would be known, subsequent analysis of repair and alteration would be facilitated.