Zhang H, Herman P L, Weeks D P
Biochemistry Department, University of Nebraska, Lincoln 68583-0718.
Plant Mol Biol. 1994 Feb;24(4):663-72. doi: 10.1007/BF00023562.
Hundreds of mutants with defects in a variety of physiologically important functions, such as photosynthesis, respiration, flagellar motility, phototaxis, circadian rhythms and the cell cycle, have been isolated from cultures of Chlamydomonas reinhardtii. In only a few cases have the genes responsible for these mutations been cloned and sequenced. The development of efficient methods for transformation with nuclear genes [7] has allowed the recent demonstration of gene isolation through genomic complementation with a pooled library of C. reinhardtii DNA [9]. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed (ordered) cosmid library of 11,280 individual clones contained in the separate wells of 120 microtiter plates. The average insert size is ca. 38 kb. PCR analysis of five sequenced nuclear genes present in the Chlamydomonas library revealed a range from two copies for the alpha 2- and beta 2-tubulin genes to at least seven copies for the argininosuccinate lyase gene. Overall, these five clones were represented an average of > or = 3.4 times in the library. Thus, the probability that any one particular nuclear gene of < 1000 bp will be found in the library is > or = 97%, and the probability that a gene of ca. 10,000 bp will be found in the library is ca. 92%. Rapid screening methods with cosmid DNAs pooled from individual microtiter dishes have been applied successfully. Bacteria containing clones of the argininosuccinate lyase gene have been identified through genomic complementation of a Chlamydomonas mutant bearing an inactive argininosuccinate lyase gene.(ABSTRACT TRUNCATED AT 250 WORDS)
从莱茵衣藻培养物中已分离出数百种在各种生理重要功能方面存在缺陷的突变体，这些功能包括光合作用、呼吸作用、鞭毛运动、趋光性、昼夜节律和细胞周期等。只有少数情况下，导致这些突变的基因被克隆和测序。核基因转化高效方法的发展[7]使得最近通过用莱茵衣藻DNA混合文库进行基因组互补来分离基因成为可能[9]。为了提高分离与特定突变互补基因的效率，我们建立了一个索引（有序）黏粒文库，该文库包含11280个单独克隆，分布在120个微量滴定板的不同孔中。平均插入片段大小约为38 kb。对莱茵衣藻文库中存在的五个已测序核基因进行PCR分析发现，α2 - 和β2 - 微管蛋白基因有两个拷贝，而精氨琥珀酸裂解酶基因至少有七个拷贝。总体而言，这五个克隆在文库中的平均代表倍数≥3.4倍。因此，在文库中找到任何一个小于1000 bp的特定核基因的概率≥97%，找到一个约10000 bp基因的概率约为92%。从单个微量滴定板中汇集黏粒DNA的快速筛选方法已成功应用。通过对携带无活性精氨琥珀酸裂解酶基因的莱茵衣藻突变体进行基因组互补，已鉴定出含有精氨琥珀酸裂解酶基因克隆的细菌。（摘要截短至250字）
