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Applied and Environmental Microbiology, September 2004, p. 5298-5304, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5298-5304.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Modified Serial Analysis of Gene Expression Method for Construction of Gene Expression Profiles of Microbial Eukaryotic Species

Kathryn J. Coyne,¹ JoAnn M. Burkholder,² Robert A. Feldman,^3, David A. Hutchins,¹ and S. Craig Cary^1*

Graduate College of Marine Studies, University of Delaware, Lewes, Delaware,¹ Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, North Carolina,² Amersham Biosciences, Inc., Sunnyvale, California³

Received 16 February 2004/ Accepted 13 May 2004

Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3' cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.

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* Corresponding author. Mailing address: University of Delaware Graduate College of Marine Studies, 700 Pilottown Rd., Lewes, DE 19958. Phone: (302) 645-4288. Fax: (302) 645-4007. E-mail: caryc{at}udel.edu.

ECOHAB contribution number 102.

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Applied and Environmental Microbiology, September 2004, p. 5298-5304, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5298-5304.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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