Lactobacillus-mediated RNA interference in nematode.

We engineered Lactobacillus paracasei to produce a dsRNA that would trigger RNAi-induced silencing of an essential gene in the nematode Caenorhabditis elegans. The dsRNA-expressing L. paracasei can be used in experiments conducted on culture plates and may also be used as an orally administrable dsRNA carrier for humans and other mammals.

Transcription factors GATA/ELT-2 and forkhead/HNF-3/PHA-4 regulate the tropomyosin gene expression in the pharynx and intestine of Caenorhabditis elegans.

Gene regulation during development is an important biological activity that leads to synthesis of biomolecules at specific locations and specific times. The single tropomyosin gene of Caenorhabditis elegans, tmy-1/lev-11, produces four isoforms of protein: two from the external promoter and two from the internal promoter. We investigated the internal promoter of tropomyosin to identify sequences that regulate expression of tmy-1 in the pharynx and intestine. By promoter deletion of tmy-1 reporters as well as by database analyses, a 100-bp fragment that contained binding sequences for a GATA factor, for a chicken CdxA homolog, and for a forkhead factor was identified. Both the forkhead and CdxA binding sequences contributed to pharyngeal and intestinal expression. In addition, the GATA site also influenced intestinal expression of tmy-1 reporter. We showed that ELT-2 and PHA-4 proteins interact directly with the GATA and forkhead binding sequences, respectively, in gel mobility shift assays. RNA interference knockdown of elt-2 diminished tmy-1::gfp expression in the intestine. In contrast to RNA interference knockdown of pha-4, expression of tmy-1::gfp in pha-4;smg-1 mutants was slightly weaker than that of the wild type. Ectopic expression of PHA-4 and ELT-2 by heat shock was sufficient to elicit widespread expression of tmy-1::lacZ reporter in embryos. We found no indication of a synergistic relation between ELT-2 and PHA-4. Based on our data, PHA-4 and CdxA function as general transcription factors for pharyngeal and intestinal regulation of tmy-1. We present models by which ELT-2, PHA-4, and CdxA orchestrate expression from the internal promoter of tmy-1.

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: the role of the C- and N-terminal extensions.

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.

Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans.

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni-1, tni-2/unc-27 and tni-3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni-4 was expressed solely in the pharynx. Expressing tni-1 and -2 gene RNAi caused motility defects similar to unc-27 (e155) mutant, a tni-2 null allele. The tni-3 RNAi expression produced egg laying defects while the tni-4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI-4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

Molecular mechanisms and drug development in aquaporin water channel diseases: aquaporin superfamily (superaquaporins): expansion of aquaporins restricted to multicellular organisms.

Eleven aquaporins are identified in mammals. They all have highly conserved two asparagine-proline-alanine (NPA) boxes that are important for the formation of the water permeating pore. Recently we identified two new proteins in mammals distantly related to aquaporins (AQPs) with poorly conserved NPA boxes. Similarly poorly conserved AQP-like proteins were found in several genome projects of multicellular organisms. They may be subgrouped as the AQP superfamily or superaquaporins. Their function is still unknown. AQP11 knockout mice suffer from polycystic kidneys and neonatal fatality. The identification of superaquaporins will provide new insights into the role of AQPs in organogenesis.

Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans.

The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca(2+) binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.