Harper J F, Huson K S, Kirk D L
Department of Biology, Washington University, St. Louis, Missouri 63130.
Genes Dev. 1987 Aug;1(6):573-84. doi: 10.1101/gad.1.6.573.
The regA locus plays a centrally important role in Volvox development by preventing somatic cells from redifferentiating as germ cells; until now, approaches to cloning regA, as a preliminary to molecular analysis of its function, have been lacking. Here a novel approach is described that uses repetitive-sequence probes to rapidly identify restriction fragment length polymorphisms (RFLPs) linked to regA. Genomic DNA was cut with restriction enzymes having 4-base recognition sequences and then electrophoresed long enough to run most fragments off the gel; the remaining long (1- to 20-kb) fragments were resolved into numerous, reproducibly identifiable bands. On Southern blots of such preparations, six repetitive-sequence probes were used to identify 1232 bands, 24% of which were polymorphic between two closely related strains. Ninety-four RFLPs, for which inheritance patterns have been analyzed, fall into 36 "segregation groups," within which no recombination was observed in the limited progeny sample analyzed. Eight RFLPs cosegregated perfectly with alleles at the mating-type (mt) locus. More significantly, four RFLPs exhibited linkage to the regA locus, providing a potential starting place for a chromosome walk designed to clone the locus.
regA基因座在团藻发育过程中起着至关重要的核心作用，它能阻止体细胞再分化为生殖细胞；到目前为止，一直缺乏克隆regA的方法，而这是对其功能进行分子分析的前提。本文描述了一种新方法，该方法利用重复序列探针快速鉴定与regA连锁的限制性片段长度多态性（RFLP）。用具有4碱基识别序列的限制性内切酶切割基因组DNA，然后进行足够长时间的电泳，使大多数片段跑出凝胶；剩余的长片段（1至20 kb）被分离成许多可重复识别的条带。在这类制备物的Southern杂交印迹上，使用六种重复序列探针鉴定出1232条带，其中24%在两个密切相关的菌株之间具有多态性。已分析其遗传模式的94个RFLP属于36个“分离组”，在分析的有限后代样本中，组内未观察到重组现象。八个RFLP与交配型（mt）基因座的等位基因完全共分离。更重要的是，四个RFLP与regA基因座表现出连锁关系，为旨在克隆该基因座的染色体步移提供了一个潜在的起始点。
