METHODS AND PLATFORMS 



Laboratory Studies . To pave the way for studies with organisms that are more difficult to culture 

 and extract, we have begun our investigations with marine cyanobacterial strains with which we 

 have some experience. Anabaena sp. strain CA is a marine nitrogen fixing organisms that we 

 isolated, along with several other strains, 17 years ago. It is representative of a group of marine 

 cyanobacteria found on the ocean margins and it grows well in the lab. We have worked out all 

 the basic techniques for measuring RubisCO transcripts from this organism under both light and 

 nutrient limitation. These studies resulted in the significant observation that the RubisCO genes 

 (rbcLrbcS) and the gene {red) that specifies an enzyme that modulates RubisCO activity in vivo, 

 namely RubisCO activase, are independently regulated at the level of transcription. These studies 

 are relevant to understanding the ability of RubisCO to fix C0 2 which drives the ecosystems of 

 the ocean margins. 



For the study of RubisCO regulation, four types of biological measurements are required: 

 1) measurement of transcriptional regulation, by extraction and quantitation of rbcL mRNA 2) 

 measurement of RubisCO enzyme activity in extracts of cells 3) determination of the amount of 

 RubisCO protein, determined immunologically and 4) determination of whole cell carbon 

 fixation. These methods provide closure on all mechanisms of regulation for this enzyme. 

 Additionally, we will amplify, clone, and sequence some rbcL genes from natural populations of 

 phytoplankton and from specific cultures provided to us by collaborators and of our own 

 isolation. 



For the studies described herein, approximately 20x20' deck space is required. A vessel 

 with a CTD with a fluorometer probe, light meter, and rosette sampler is required. In terms of 

 shipboard lab facilities, 2 fume hoods are required, one for mRNA extraction, one for filtration 

 of samples treated with DEPC (diethyl pyrocarbonate). Adequate space for radioisotope work is 

 also required. Deck space for incubators is also needed. For these studies to be performed in 

 conjunction with other components of the DOE OMP, a reasonably large vessel will be required. 



STRENGTHS AND LIMITATIONS OF PROPOSED RESEARCH 



Strengths. Clearly one of the greatest strength of this project is the capability of measuring 

 phytoplankton gene expression by mRNA isolation and quantitation. A second strength is the 

 amplification of rbcL genes for cloning and sequencing, which will enable determination of the 

 types of organisms responsible for oceanic carbon fixation. 



Limitations. A potential limitation of the project deals with uncertainty on the appropriateness 

 of our cyanobacterial gene probe to detect oceanic picoplankton rbcL genes. Although we have 

 had a high degree of success using the A. nidulans (= Synechococcus PCC6301) probe, some 

 recent studies in the Tabita lab indicate that it only weakly hybridizes with Synechococcus 

 WH7803 (although this organism may not be representative of oceanic picocyanobacteria). Our 

 preliminary studies on the Pacific Prochlorococcus by sequence analysis suggests that it would 



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