GENE ISOLATION THROUGH GENOMIC COMPLEMENTATION USING AN INDEXED LIBRARY OF CHLAMYDOMONAS-REINHARDTII DNA

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 agininosuccinate lyase gene. Overall, these five clones were represented an average of greater than or equal to 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 greater than or equal to 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 arginnosuccinate lyase gene. We are using the nomenclature of'indexed' library versus 'ordered' library to avoid confusion of this library with a library of ordered contigs.