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Research funded by the Joint NSF-USDA Initiative
Microbial Genome Sequencing Project
 

An EST Approach to Understanding Endosymbiotic
Gene Transfer (NSF MCB 02-36631)
  
 
PI:
Debashish Bhattacharya
  Co-PI: M. Bento Soares
 
PROJECT SUMMARY:
Eukaryotic endosymbiosis, in which a single-celled protist engulfs and "enslaves" the photosynthetic organelle (plastid) of another cell, explains the origin of most algae. Plastid endosymbiosis necessarily changes the "host" lineage because of the large scale lateral transfer of genes from the endosymbiont to the host nuclear genome that is required to ensure proper organelle function. A genome-wide approach is necessary to address this poorly understood process. We propose to sequence 30,000 expressed sequence tags (ESTs) from the dinoflagellate, Alexandrium tamarense, and from the source of its plastid, a haptophyte, Emiliania huxleyi. High-quality non-normalized, normalized and serially subtracted cDNA libraries will be constructed using mRNA extracted from unialgal cultures. These libraries will be used in a highly efficient gene discovery strategy for generation of a comprehensive collection of algal cDNAs. This strategy relies on the generation of ESTs from serially subtracted normalized libraries and has yielded unprecedented rates of gene discovery in three large-scale programs currently being conducted at the University of Iowa. Using these techniques we will produce about 10,000 unique ESTs from each algal species. These data will be the basis for understanding the extent of gene transfer from the endosymbiont to the host and will provide other scientists with an invaluable resource for understanding the biology of two taxa that are of high economic and ecological importance. Alexandrium causes harmful algal blooms in coastal areas resulting in paralytic shellfish poisoning whereas Emiliania is dominant in oceanic open waters where its coccolithophore phase acts as a major carbon sink on our planet.		  
 

STATEMENT OF WORK
  
Aim 1.To construct individually tagged non-normalized and normalized cDNA libraries from the dinoflagellate alga, Alexandrium tamarense, and the haptophyte alga, Emiliania huxleyi.  
 

Starter libraries will be constructed from exponentially growing uni-algal cultures of Amphidinium and Emiliania (available from the CCMP culture collection). A total of two non-normalized and two normalized libraries will be constructed from algal cDNA. Each library will be tagged with a unique 5 bp identifier, which makes identification of the source organism readily apparent.

  

Aim 2. To generate serially subtracted normalized libraries increasingly enriched for the mRNAs of the complex frequency class (rare mRNAs). The subtracted libraries will be derived from a very complex starting library mixture comprising all normalized libraries constructed in specific aim 1, by an iterative process that we have developed and named Serial Subtraction of Normalized Libraries (Bonaldo, Lennon, and Soares 1996, Soares 1997). 		 
 

Serial Subtraction of Normalized Libraries is an iterative process whereby each new set of arrayed cDNA clones is pooled and subtracted from the library in which they originated. As a result, each new subtracted library of a series is progressively enriched for novel cDNAs (i.e., cDNAs from which ESTs have not been generated yet). This strategy was specifically developed to expedite gene discovery in EST programs. It minimizes redundant identification of the most common ESTs while enhancing the representation of the rarest cDNAs, which are less likely to be sampled in gene discovery programs that rely solely on random picking of clones from standard libraries.

 

Aim 3. To submit to GenBank 30,000 3’ ESTs derived from the serially subtracted libraries constructed in specific aim 2, and to conduct clustering analyses of the EST data to identify up to 20,000 unique ESTs (about 10,000 from each species [based on an expected 66% discovery rate]). At a minimum, we expect to isolate 12,000 unique ESTs from the 30,000 sequences. These predictions are realistic goals based on our (B. Soares lab) extensive experience utilizing serial subtraction for a rat EST discovery project (Sheetz et al. 2001, see http://ratest.eng.uiowa.edu/). 		 

Aim 4. To make all sequence data, clones, and libraries promptly available for wide use in the scientific community. All validated sequences will be annotated and submitted to GenBank (dbEST) on a quarterly basis. 		 
 
 
 
 
Algal Genomics: Year one activities Year two activitiesStudy organismslink to EST site