Sylvia Y M Yao, David Coates, Eugene G Chomey, R Elwyn Isaac, Mark Griffiths, Darryl MacGregor, Ian A Hope, Peter J Appleford, Stephen A Baldwin, James D Young, Carol E Cass
[
International C. elegans Meeting,
2001]
Nucleoside transporters (NTs) are essential for nucleotide synthesis by salvage pathways in cells, such as protozoan parasites, that lack de novo biosynthetic pathways. In addition, they have important roles in many adenosine-mediated physiological processes in mammals, including neurotransmission, platelet aggregation and coronary vasodilation. Most eukaryotes possess multiple NTs, which can be grouped into two unrelated transporter families; the equilibrative nucleoside transporters (ENTs) and the concentrative transporters (CNTs). In order to understand the physiological functions of nucleoside transporters and the reasons underlying their biological diversity, we are using C. elegans as a model system. C. elegans possesses two genes encoding CNTs and six encoding putative ENTs. As the first step towards understanding the rationale underlying this diversity of transporters, we have expressed one of these, ZK809.4, in Xenopus oocytes and shown it to be a genuine ENT which we have designated CeENT1. It exhibits a broad substrate specificity for natural purine and pyrimidine nucleosides and also transports antiviral nucleoside analogues such as 3'-azido-3'-deoxythymidine (AZT). cDNAs for two other ENT genes K09A9.3 and K02E11.1 (which we have designated CeENT2 and CeENT3 respectively) have been obtained from Yuji Kohara and have also been expressed in Xenopus oocytes. We have demonstrated that these are able to transport nucleosides, with adenosine being a good substrate for CeENT2 and adenosine and uridine being substrates for CeENT3. In an attempt to elucidate the biological roles of these and other NTs, we have used Green Fluorescent Protein reporter constructs to investigate the temporal-spatial expression patterns of these genes. Additional clues to the biological roles of NTs have been obtained by double-stranded RNA interference (dsRNAi). RNAi using dsRNA corresponding to CeENT1 failed to yield a discernible phenotype. Similarly, injection of animals with dsRNA corresponding to CeENT2 yielded no obvious phenotype. However, the progeny of worms co-injected with a mixture of CeENT1 and CeENT2 RNAs exhibited an everted vulval phenotype, suggesting that CeENT1 and CeENT2 exhibit a redundant function.
James D Young, Sylvia Y M Yao, Carol E Cass, David Coates, Stephen A Baldwin, Darryl MacGregor, Peter J Appleford, Eugene G Chomey, Ian A Hope, Mark Griffiths, R Elwyn Isaac
[
European Worm Meeting,
2002]
Nucleoside transporters (NTs) are essential in cells that lack de novo biosynthetic pathways. In addition, they have important roles in many adenosine-mediated physiological processes in mammals, including platelet aggregation and coronary vasodilation. Most eukaryotes possess multiple NTs, which can be grouped into two unrelated transporter families; the equilibrative nucleoside transporters (ENTs) and the concentrative transporters (CNTs). In order to understand the physiological functions of nucleoside transporters and the reasons underlying their biological diversity, we are using C. elegans as a model system. C. elegans possesses two genes encoding CNTs and six encoding putative ENTs. As the first step towards understanding the rationale underlying this diversity of transporters, we expressed one of these, ZK809.4, in Xenopus oocytes and showed it to be a genuine ENT which we have designated CeENT1. It exhibits a broad substrate specificity for natural purine and pyrimidine nucleosides and also transports antiviral nucleoside analogues such as 3'-azido-3'- deoxythymidine (AZT). cDNAs for two other ENT genes, K09A9.3 and K02E11.1 (which we have designated CeENT2 and CeENT3 respectively) have been obtained from Yuji Kohara and have also been expressed in Xenopus oocytes. We have demonstrated that these are able to transport nucleosides, with adenosine being a good substrate for CeENT2 and adenosine and uridine being substrates for CeENT3. We have generated a further two cDNAs for the ENT genes F16H11.3 and C47A4.2 (designated CeENT4 and CeENT5 respectively). CeENT5 has been expressed in Xenopus oocytes and is able to transport a variety of substrates. We have also demonstrated that transport of uridine is affected by the inhibitor dipyridamole, but not by draflazine or dilazep. We have used Green Fluorescent Protein reporter constructs to investigate the temporal-spatial expression patterns of NT genes. GFP expression of these reporters is observed principally in gut cells, although expression of some constructs was seen in other cells, such as the pharynx and vulval muscle cells. Additional clues to the biological roles of NTs have been obtained by double-stranded RNA interference (dsRNAi) and by obtaining deletion mutants from the C. elegans knockout consortium. RNAi experiments using dsRNA corresponding to CeENT1 and CeENT2 give rise to worms with an everted vulva and our experiments suggest that these two genes are co-redundant. Deletion mutants for CeENT2 and CeENT4 have been obtained from the knockout consortium and although the CeENT2 mutant appears to have no vulval phenotype, brood size is affected. Injection of CeENT2 deletion mutants with CeENT1 dsRNA also phenocopies the affect seen using CeENT1 and CeENT2 dsRNAs together. CeENT4 deletion worms show a severe phenotype, with everted vulva and vastly reduced brood sizes. These results suggest that at least 3 of the 6 ENTs are required in the worm for proper growth and development.