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Published on January 23, 2003; 10.1104/pp.016899

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Received October 29, 2002
Returned for revision November 13, 2002
Accepted November 19, 2002

Chlamydomonas reinhardtii Genome Project. A Guide to the Generation and Use of the cDNA Information

Jeff Shrager *, Charles Hauser , Chiung-Wen Chang , Elizabeth H. Harris , John Davies , Jeff McDermott , Raquel Tamse , Zhaodou Zhang , and Arthur R Grossman

Department of Plant Biology, The Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305 (J.S., C.-W.C., Z.Z., A.R.G.); Biology Department, Duke University, DCMB Box 91000, Durham, North Carolina 27708 (C.H., E.H.H.); Department of Botany, Iowa State University, 353 Bessey Hall, Ames, Iowa, 50011 (J.D., J.M.); and Stanford Genome Technology Center, 855 California Avenue, Palo Alto, California 94304 (R.T.)

* Corresponding author; email: jshrager{at}andrew2.Stanford.edu.

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.




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