Berns M W
Science. 1974 Nov 22;186(4165):700-5. doi: 10.1126/science.186.4165.700.
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-micro.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 PTK(2) 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.
在本文中，我展示了一些数据，这些数据表明实现引言中概述的五个目标是可行的。通过以下方法应该能够将基因定位到特定的染色体区域：（i）选择性地删除一条或两条同源染色体上0.25至0.5微米的DNA片段；（ii）删除一条或两条完整的同源染色体；或（iii）将细胞融合与全染色体的选择性删除相结合，然后再删除染色体片段。通过激光微照射，应该能够通过从单个细胞中去除全染色体以及核型中每条染色体的染色体片段，然后评估每个细胞的克隆效率，来确定哪些染色体和染色体区域对细胞的即时存活至关重要。例如，我们已经确定从PTK(2)细胞中去除一条大的1号染色体并不妨碍细胞进行随后的有丝分裂。通过用激光束损伤选定的小面积DNA，然后克隆受照射的细胞，也应该能够产生新类型的突变体——但这还有待证明。这个过程可能会揭示未受照射同源染色体上的隐性等位基因，或者可能导致直接产生基因突变。对两条同源染色体上相同位置进行照射可能会导致一个基因座的缺失，最终可能会被检测为代谢途径中的缺陷或其他一些细胞异常。可以用激光照射的细胞进行染色体稳定性和DNA恒定性的研究。例如，对1号染色体的核型分析表明，存在一种细胞机制来维持该染色体在二倍体和四倍体细胞系中的恒定性。核型中的每条染色体都可以采用相同的方法。可以使用各种细胞化学程序对细胞进行DNA定量研究，并且可以在激光微照射后的不同时间进行染色体和DNA分析。还可能研究激光照射引起的染色体损伤的修复。可以在照射后的不同时间通过放射自显影、细胞化学和电子显微镜程序检查细胞，并且由于突变事件的精确位置、时间和性质是已知的，这将便于后续对修复和改变的分析。
