The Caenorhabditis elegans K10C2.4 gene encodes a member of the fumarylacetoacetate hydrolase family: a Caenorhabditis elegans model of type I tyrosinemia.

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.

A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.

BACKGROUND: The nematode Caenorhabditis elegans has emerged as a powerful system to study biologic questions ranging from development to aging. The generation of transgenic animals is an important experimental tool and allows use of GFP fusion proteins to study the expression of genes of interest or generation of epitope tagged versions of specific genes. Transgenes are often generated by placing a promoter upstream of a reporter gene or cDNA. This often produces a representative expression pattern, but important exceptions have been observed. To better capture the genuine expression pattern and timing, several investigators have modified large pieces of DNA carried by BACs or fosmids for use in the construction of transgenic animals via recombineering. However, these techniques are not in widespread use despite the advantages when compared to traditional approaches. Additionally, some groups have encountered problems with employing these techniques. Hence, we sought identify ways to improve the simplicity and reliability of the procedure. RESULTS: We describe here several important modifications we have made to existing protocols to make the procedure simpler and more robust. Among these are the use of galK gene as a selection marker for both the positive and negative selection steps in recombineering, the use of R6K based plasmids which eliminate the need for extensive PCR product purification, a means to integrate the unc-119 marker on to the fosmid backbone, and placement of homology arms to commonly used GFP and TAP fusion genes flanking the galK cassette which reduces the cost of oligos by 50%. CONCLUSION: We have made several significant changes that allow the production of C. elegans transgenes from a commercially available fosmid library in a robust and streamlined manner. These changes make the technique more attractive especially to small academic labs unfamiliar with recombineering.