The aim of this R21 proposal is to establish the nematode Caenorhabditis elegans (C. elegans) as a model organism to further study the mechanisms and regulation of intestinal transport of the water-soluble vitamins at the integrated whole animal level in vivo. The water-soluble vitamins are essential for normal cellular functions and growth, and their deficiencies lead to a variety of clinical abnormalities. Humans cannot synthesize these micronutrients, and thus, must obtain them by intestinal absorption via the function of a number of specialized transporters. Knowledge about these transport systems has been forthcoming in recent years, but mainly through the use of reductionist in vitro approaches in mammalian systems. At the whole animal level in vivo, however, much less is currently known. Such knowledge could be obtained if a suitable animal model system is available. We propose that the nematode C. elegans represents such an animal model, and plan to establish it in our laboratory. This animal model is anatomically simple, transparent, easy to manipulate, has a defined genome, and has a relatively short life span. In addition, a majority of human genes appear to have orthologs in this nematode. To begin our effort, we have searched the C. elegans genome and have identified three putative orthologs to the human reduced folate carrier (hRFC), i.e., ceRFC- 1, ceRFC-2 and ceRFC-3. The putative ceRFC-1 showed the highest degree of identity with the hRFC, and thus, was cloned and shown by functional expression to be indeed a folate transporter. We have also cloned the full-length 5'-regulatory region of the ceRFC-1 gene (approx. 1.5 kb), fused it to the Firefly luciferase reporter gene, and confirmed its promoter activity. In addition, we have generated a fusion transcriptional construct of the same ceRFC-1 5 '-regulatory region with the green fluorescent protein (GFP), i.e., ceRFC1- GFP, and showed its expression in the intestine. To achieve our goal of establishing the C. elegans model, we propose the following specific aims: 1) To further characterize the cloned ceRFC-1, 2) To determine the pattern of ceRFC-1 expression in the intestine at different developmental stages of C. elegans, 3) To identify the minimal promoter region that dictates expression of this gene in the intestine and the cis-regulatory elements involved, 4) To examine potential adaptive regulation in the expression of the ceRFC-1 in the intestine in response to changes in extra-cellular folate availability, and 5) To examine the effect of silencing the ceRFC-1 gene by RNA interference (RNAi) on the C. elegans phenotype, reproduction, survival, and life span. The successful completion of these studies should allow us to establish the C. elegans model and make us familiar with its utilities. This should be of great value in future investigations into the mechanisms and regulation of transport of water-soluble vitamins in the intestine (and other tissues) at the integrated whole animal level in vivo. ? ? ?
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| Balamurugan, Krishnaswamy; Ashokkumar, Balasubramaniem; Moussaif, Mustapha et al. (2007) Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans. Am J Physiol Cell Physiol 293:C670-81 |
