ISOLATION AND GENETIC ANALYSIS OF MUTANT STRAINS OF CHLAMYDOMONAS REZNHARDZ DEFECTIVE IN GAMETIC DIFFERENTIATION URSULA W. GOODENOUGH, CAROL HWANG AND HOWARD MARTIN The Biological Laboratories, Harvard Uniuersiiy, Cambridge, Massachusetts 02138 Manuscript received February 10, 1975 Revised copy received October 22, 1975 ABSTRACT Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-6, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or mt+ gametes; these strains have been induced to form rare zygotes with mt- gametes and the mutations are shown to be unlinked to the mi locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-I strain agglutinate well with mt gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that imp-I gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amen- able to genetic dissection. HE unicellular biflagellate green alga Chlamydomonas reinhardi is often Tcited as representing the kind of eukaryotic protist that once gave rise to the multicellular plants and animals: it possesses all of the “standard” organelles found in metazoan cells (SAGER and PALADE 1957; RINGO 1967; JOHNSON and PORTER 1968), but it has not acquired the exotic structural features found in the more highly evolved protozoa; its chloroplast membrane polypeptides (LEVINE and DURAM 1973) and chloroplast functions (LEVINE 1968) are similar to a higher plant such as spinach; its chromatin structure and mitotic behavior resemble higher organisms and do not exhibit the “deviations” found in such organisms as yeast (MOENS and RAPPORT 1971) or dinoflagellates (KUBAI and RIS 1969); and its haploid DNA content [0.1 picogram (CHIANG and SUEOKA 1967)] is of the same order of magnitude as an organism like Drosophila r0.25 picogram (MULDER, VAN DUISIN and GLOOR 1968)l. If such a central evdu- tionary position is supposed, then it becomes of considerable interest to investigate the genetic and biochemical capacity of C. reinhardi to carry out cellular differ- entiation and to establish cell-to-cell contacts, both kinds of processes being clearly critical to metazoan existence. Genetics 82: 169-186 February, 1976