RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE GENE-EXPRESSION AND DIVERSITY OF LAKE-ERIE PLANKTONIC MICROORGANISMS/

Carbon dioxide fixation is carried out primarily through the Calvin-Be nson-Bassham reductive pentose phosphate cycle, in which ribulose-1,5- bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme, The pr imary structure of the large subunit of form I RubisCO is well conserv ed; however, four distinct types, A, B, C, and D, may be distinguished , with types A and B and types C and D more closely related to one ano ther, To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA a nd DNA from four Lake Erie sampling sites, Probes prepared from RubisC O large-subunit genes (rbcL) of the freshwater cyanobacterium Synechoc occus sp, strain PCC6301 (representative of type IB) and the diatom Cy lindrotheca sp, strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA, To quantitate rbcL, gene expression for e ach sample, the amount of gene expression per gene dose (i,e., the amo unt of mRNA divided by the amount of target DNA) was determined, With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shi fted toward open water, By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose, Compl ementary DNA specific for rbcL was synthesized from Lake Erie RNA samp les and used as a template for PCR amplification of portions of variou s rbcL genes. Thus far, a total of 21 clones of rbcL genes derived fro m mRNA have been obtained and completely sequenced from the Ballast Is land site, For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae, In co ntrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages, At 5 m deep, the active CO2- fixing planktonic organisms represented a diverse group, including org anisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse site s must be accomplished, the discovery of distinctly different sequence s of rbcL mRNA at different water depths suggests that there is a stra tification of active CO2-fixing organisms in western Lake Erie.