It is now known that silicon (Si) is an essential trace element in animals for the formation of bone, cartilage, and connective tissue; it participates in female hormonal balance (in rats) and is a pathogenic agent in fibrotic lung diseases and cancers. It is interrelated with calcium and hormones in atherosclerosis and aging, and with aluminum in Alzheimer's disease. Though Si is clearly implicated in a variety of health-related questions, relatively little is known of its mode of action in mammals, due in part to inherent methodological difficulties. Many of these obstacles can be avoided by using the diatom as an experimental system, due to this organism's absolute dependence in Si. In the diatom, Si is required not only for cell wall formatin, but also for DNA synthesis and for net synthesis of cAMP. Si induces a number of mRNAs and proteins, including two nuclear DNA polymerases and thymidylate kinase, whose appearance coincides with DNA synthesis. Si may thus be a major regulator of diatom proliferation at the level of gene expression. The long term objective of our research is to understand how Si affects cellular metabolism in the diatom and other organisms; in particular whether it regulates gene expression through direct interaction with a regulatory molecule or indirectly by altering cellular metabolism. The specific goal of the proposed research is to characterize gene sequences which are responsive, either through direct or indirect effects, to Si. This will be accomplished in three studies, using Cylindrotheca fusiformis. In Study I, - """"""""Determination of the Si-responsive Regulatory Point in Gene Expression"""""""" - (1) cDNA libraries representing the distinctive mRNA populations from Si-starved and Si-replenished diatoms will be screened to identify cDNA clones derived from genes with Si-responsive mRNAs; (2) cDNA clones will be identified from particualr genes whose gene products, thymidylate kinase and nuclear DNA polymerases, are Si-responsive; (3) these cDNAs will be sequenced; and (4) hybridization probes derived from these clones will be used to determine the points in gene expression and the point(s) in the cell cycle at which Si-responsive genes are regulated. In Study II, -""""""""Development of a Diatom Transformation System"""""""" - a transformation system will be developed to re-introduce Si-responsive genes into the diatom. Based on the foundation laid in Studies I and II, in Study III - """"""""Determination of Diatom Si-responsive Regulatory Sequences"""""""" - specific Si-responsive regulatory sequences within Si-responsive genes will be identified, by fusing putative regulatory sequences appropriately to an assayable marker gene, transforming diatoms with these fusions, and determining the response of the marker gene to Si. The isolation of such sequences is an essential step in elucidating the mechanism(s) involved in Si regulation of gene expression and cell proliferation. In view of Si's importance both as a metabolite and as a pathogen, a contribution to understanding its mode of action at the molecular level is most germane to health related research.
