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 



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