Chlamydomonas is a haploid, unicellular green alga, about 10 µm in diameter. Synchronous cultures can be grown phototrophically in a simple defined medium. In addition to the two 10–12 µm-long flagella that emanate from one end of the cell, Chlamydomonas shares the cytology of typical higher plant and animal cells (e.g., basal bodies/centrioles, microtubules, actin filaments, chloroplasts, mitochondria, en-doplasmic reticulum, and Golgi). The genetics of Chlamydomonas is similar to that of yeast, with plus and minus cells mating to form zygotes, followed by meiosis and tetrad formation. Many mutants are available (Harris 1989) or can be generated by classical mutagenesis procedures. Because Chlamydomonas is easily transformed, with the introduced DNA inserting randomly into the genome (Diener et al. 1990; Kindle et al. 1989), mutants can also be generated by insertional mutagenesis, allowing for the isolation of mutant genes by plasmid rescue (Tam and Lefebvre 1993). In addition, progress is being made on gene cloning by complementation (Purton and Rochaix 1994) and on the selection of homologous recombinants (Sodeinde and Kindle 1993). We are using flagellar regeneration in this green alga as a model system for studying the biogenesis of cell organelles (Johnson and Rosenbaum 1993).

In response to the primary sex determination signal, X chromosome dose, the Sex-lethal gene controls all aspects of somatic sex determination and differentiation, including X chromosome dosage compensation. Two complementary classes of mutations have been identified that differentially affect Sxl somatic functions: (1) those impairing the "early" function used to set developmental pathway choice in response to the sex determination signal and (2) those impairing "late" functions involved in maintaining the pathway choice independent of the initiating signal and/or in directing differentiation. This "early vs. late" distinction correlates with a switch in promoter utilization from SxlPe to SxlPm at the blastoderm stage and a corresponding switch from transcriptional to RNA splicing control. Here we characterize five partial-loss-of-function Sxl alleles to explore a distinction between "early vs. late" functioning of Sxl in dosage compensation. Assaying for dosage compensation during the blastoderm stage, we find that the earliest phase of the dosage compensation process is controlled by products of the early Sxl promoter, SxlPe. Hence, in addition to triggering the sexual pathway decision of cells, products derived from SxlPe also control early dosage compensation, the first manifestation of sexually dimorphic differentiation. The effects of mutant Sxl alleles on early dosage compensation are consistent with their previous categorization as early vs. late defective with respect to their effects on pathway initiation. Results reported here suggest that the dosage compensation regulatory genes currently known to function downstream of Sxl, genes known as the "male-specific lethals," do not control all aspects of dosage compensation either at the blastoderm stage or later in development. In the course of this study, we also discovered that the canonical early defective allele, Sxlf9, which is impaired in its ability to establish the female developmental pathway commitment, is likely to be defective in the stability and/or functioning of products derived from SxlPe, rather than in the ability of SxlPe to respond to the chromosomal sex determination signal.

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.