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The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rpl2 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were largely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO(2) fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms.