We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.Studies using the unicellular eukaryotic alga Chlamydomonas reinhardtii have yielded important insights into many cellular processes including photosynthesis (45, 123), flagellar assembly and motility (24,28,86,112,114,124,137,140), basal body assembly and positioning (115), gametogenesis and fertilization (35,42,167), DNA repair (109), phototaxis (49, 139), cell wall assembly (1), circadian rhythms (91, 162), and the regulation of metabolic pathways (3,14,31,50).A major strength of C. reinhardtii as an experimental system is its usefulness for genetic experiments (45,47,74). Vegetative cells are haploid, facilitating the analysis of mutant phenotypes, but stable diploid strains can be easily produced for dominance and complementation tests. Gametes can be crossed to yield diploid zygotes that sporulate to produce four products of meiosis, allowing routine tetrad analysis. Over the past 50 years, hundreds of mutations have been isolated; more than 200 genetic loci have been mapped to 17 linkage groups (28,29,45,46,52). Mutations induced by chemical or UV mutagenesis have been supplemented recently by mutations induced by transposition of one of several transposable element families in the genome (17,34,130,133) or by insertional mutagenesis (13,101,149).Insertional mutagenesis has become the favored method for generating mutations since the development of procedures for efficient transformation of the nuclear genome (59, 132). Upon transformation, plasmid DNA inserts in random positions into the nuclear genome, facilitating cloning of affected genes by using the transforming plasmid as a hybridization probe. This method of gene tagging has led to the isolation of numerous genes identified by mutation over the past several years. Despite its usefulness, the insertional-mutagenesis approach has drawbacks, including the inability to clone essential genes, difficulty in analyzing the large deletions that occur in some cases, and a limitation in the types of phenotypes that can be found by using a method that genera...

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A series of plasmids that carry overlapping segments of F DNA encoding the genes in the traB-traC interval was constructed, and a restriction enzyme map of the region was derived. Plasmids carrying deletions that had been introduced at an HpaI site within this interval were also isolated. The ability of these plasmids to complement transfer of F lac plasmids carrying mutations in trail, traV, traW, and traC was analyzed. The protein products of the plasmids were labeled in UV-irradiated cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These analyses showed that the product of traV is a polypeptide that migrates with an apparent molecular weight of 21,000. It was not detected when [35S]methionine was used to label plasmid products, but was readily detected in '4C-amino acid labeling experiments. A 21,500-dalton product appeared to stem from the region assigned to traP. A 9,000-dalton product was found to stem from a locus, named traR, that is located between traV and traC. No traW activity could be detected from the region of tra DNA examined. Our data also indicated that traC is located in a more promoter-proximal position than suggested on earlier maps. The plasmids constructed are expected to be useful in studies designed to identify the specific functions of the traB, -P, -V, -R, and -C products.

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