Thesis

A genomic approach to identify genes associated with calcification in Emiliania huxleyi

The aim of this study is to identify genes involved in calcification and coccolith production in Emiliania huxleyi using Suppressive Subtractive Hybridization (SSH) and microarray analysis techniques. Suppressive subtractive hybridization detected 168 differentially expressed transcripts in comparing lithforming cells grown in phosphate-limited (f/50) media and non-lith forming cells grown in phosphate-replete media (f/2). The SSH library of differentially expressed genes in lith-forming cells is comprised predominately of transcripts showing no significant matches to sequences in GenBank as a function of BLAST X homology searches. A number of transcripts from the SSH library of differentially expressed genes in non-lith forming cells showed significant homology to genes encoding ribosomal proteins and RNA polymerase subunits. To validate these differentially expressed genes, cDNA microarray analysis was employed. SSH library clones along with 188 cDNA clones previously identified as being differentially expressed by microarrays, were printed as targets on glass slides and hybridized to RNA probe molecules extracted from lith and non-lith forming cells. In this study, preliminary results indicated 80 differentially expressed genes (22.5%) out of a total of344 target clones. Among the 188 up/down regulated genes from previous microarray study, 59 genes were validated (31 %), and among 156 SSH differentially expressed genes 21 were validated (13.5%).

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