Kohara Y et al. (1994) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Sugimoto A, Motohashi T, Tabara H, Watanabe H, Kohara Y

[Worm Breeder's Gazette, 1994]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan. e-mail: ykohara@lddbj.nig.ac.jp

Yasumi Ohshima et al. (2006) East Asia C. elegans Meeting "Food, feeding, digestion and defecation of the worm"

Yasumi Ohshima, Yusuke Kaku, Yuta Yoshidome, Yuji Osako, Katsuhiro Kunigo, Akiko Nakasone, Kohji Miyahara, Tomonori Nakamura

[East Asia C. elegans Meeting, 2006]

Food or nutrition is vital for the life of C. elegans as for other animals, and should affect growth, body size and activity of the worm. In the laboratory, worms usually feed on OP50, a uracil-requiring strain of E. coli, which is a good food for worms in the sense that it supports rapid growth and production of many progeny. However, E. coli is an enteric bacterium that may not be abundant in soil which is said to be the natural habitat of the worm. Avery & Shtonda (2003) identified from soil samples more than 10 bacterial species that supported growth of the worm. Yet, the entire image of the natural food of the worm is not clear. Although the mechanisms of feeding and defecation of E. coli have been extensively studied, much is still to be solved, for example, the mechanisms of food recognition, rate and target of defecation. Furthermore, virtually nothing is known on the mechanisms of digestion of food and absorption of nutrients in the gut of the worm. In mammals, many factors which seem to be involved in digestion or absorption are known, but there is little genetics in any animal. Thus, we have begun studies on some of these, and plan to ask further, as follows. 1) Heat-killed E. coli is not likely to support growth of C. elegans. 2) We are studying whether selected microorganisms are good food or not. For example, yeast S. cerevisiae probably does not support growth to adults. 3) We are trying to identify live or dead microorganisms in the gut of marked worms that were put into a soil sample, by DNA sequencing. 4) Several artificial microspheres of various size or chemical nature are used to get clues to food recognition, feeding and defecation. 5) We are trying to analyze the time course of feeding, digestion and defecation of GFP-marked E. coli. 6) We have found many C. elegans genes possibly involved in digestion or absorption based on amino acid sequence homology with those in mammals. We have obtained KO mutants for some of them from Dr. S. Mitani for reverse genetics. 7) We have begun isolation of mutants on digestion or absorption. The screening at present uses GFP-marked E. coli as food. Two candidate mutants were obtained that retain more GFP fluorescence in the gut without little change in the pumping cycle.

A Asano et al. (1999) International C. elegans Meeting "Homologues of claudin, integral protein of mammalian tight junction, are present and important in C. elegans"

H Sasaki, A Asano, M Furuse, S Tsukita, K Asano

[International C. elegans Meeting, 1999]

Recently, new integral proteins of tight junction were discovered in mouse and human (Furuse et al., J. Cell Biol., 141, 1539, 1998; Morita et al., PNAS, 96, 511, 1999). These claudin family proteins are members of tight junction strands. Although presence of tight junctions in C. elegans is not reported, septate junctions and septate-like junctions seem to play similar functions instead. We searched the gene database of C. elegans , and found two homologues of claudin family proteins (claudin-CE1 and -CE2) with four-transmembrane domains, conserved two Cys in the first loop, and similar molecular weight. Interestingly, a protein (claudinD) was also found that has molecular weight about twice of claudin-CE1, and other characteristic structures are likely to have two claudin molecules tandemly repeated. These 3 proteins are coded from nearby sites on chromosome X. Claudin-CE1::GFP with 1.2kb upstream promoter region was expressed in spermatheca which is known to have septate junctions, and gut. Expression of claudin-CE2::GFP was much less, but tissue distribution was similar. RNAi experiments using dsRNA mixture of claudin-CE1, claudin-CE2 and Exon1-4 fragment of claudinD were performed. About 40% of F1 of the injected worms have decreased F2 production (in average 48% decrease), whereas 22% of F1 have almost normal numbers of F2's. Thus, these proteins seem to be important for reproduction of the worms. When expressed in MDCK-II epithelial cells, Claudin-CE1::GFP was localized at cell-cell junctions. Electron microscopic studies are under way. We are grateful to Miss. Akiko Kamamoto whose technical assistance make this work possible.

Kohara Y et al. (1995) Worm Breeder's Gazette "The C. elegans cDNA Project: A Progress Report."

Kohara Y, Motohashi T, Watanabe H, Sugimoto A, Tabara H

[Worm Breeder's Gazette, 1995]

The C.elegans cDNA project: A progress report Yuji Kohara, Tomoko Motohashi, Akiko Sugimoto, Hisako Watanabe and Hiroaki Tabara Gene Library Lab, National Institute of Genetics, Mishima 411, Japan e-mail: ykohara/*ddbj.nig.ac.jp Tag sequencing is now on the third set of cDNA dones. After analysis of the first set of cDNA clones (some 4,400 clones), each 10,000 clones were picked up randomly from 3 different cDNA libraries (an embryonic cDNA library and libraries of >2kb cDNA and unfractionated cDNA made from mixed stage population). The total 30,000 clones were gridded and probed with the cDNA clones belonging to the species which had been represented by more than 4 clones in the analysis of the first set. A set of some 4,800 cDNA clones (the second set) were selected out of the unhybridized clones (from rare or not analyzed cDNA species) and has been subjected to the tag sequencing. This analysis produced 3,667 clean 3'-tags which gave 1,532 more unique cDNA species (see Fig.). As the next step, the grids were further screened with the cDNA probes the groups containing more than 4 clones at the point. A set of some 4,000 cDNA clones (the third set) was selected out of the unhybridized clones and tag sequencing has been continued on this set. The current status of our progress is that we have identified 3,324 unique cDNA species out of 7,647 clones (clean 3'-tags). The unique cDNA species were assigned serial numbers from CELK00001 to CELK03324. These analyses have also detected many pairs of clones which appeared to be generated by alternative splicing. In some cases, two groups were turned out that they were derived from the same gene but had different 3'-end sequences due to alternative splicing or differential poly-A addition. We are going to make a list of such differential splicings. BLASTX search showed that 653 groups out of the 1,816 groups identified through the analysis of the second and the third sets gave significant similarities (blastx score > 100), which are listed below. (Note; "-" in the column of "Frame" means BLASTX search was made using only 3'-tag sequences so far.) Mapping and in situ analysis are in progress.

Akira ASANO et al. (2001) International C. elegans Meeting "Characterization of C. elegans claudins"

Shoichiro TSUKITA, Akira ASANO, Kimiko ASANO, Hiroyuki SASAKI, Mikio FURUSE

[International C. elegans Meeting, 2001]

Homologues of claudin, major integral protein of mammalian tight junction with four span transmembrane structure, were found in the genomic data base of C. elegans . Among them, CLC-1(C09F12.1) was discovered by a blast search with mouse claudin-6, it was found later that homology is also present with mouse claudin-7 and human claudin-14. It has in its first loop two Cys residues that are conserved among mammalian claudins. CLC-2 (T05A10.2) was found by a blast search with CLC-1, and has 33% identity (70% homology) with CLC-1, although no significant homology was found with vertebrate claudins so far known. CLC-3 (ZK563.4) has homology with mouse claudin-10 and cattle claudin-16. CLC-4 was found with a blast search with mouse claudin-6, but it was thought to be an eight span transmembrane protein at that time. Recently, it was found that this gene (C01C10.1) encodes two separate proteins, and the down-stream operon (C01C10.1b) was reported to encode a Gas3/PMP-22 homlogue (Agostoni E.et al., Gene, 234, 267-274, 1999). Product of the other operon (C01C10.1a) was not characterized well. Homology with claudin-6, -7, and -9 was found with this product, therefore named as CLC-4. These four claudin homologues have either PMP-22/EMP/MP20 motif or transmembrane four signature or both, and most of them have srg integral membrane protein motif. Furthermore, two conserved Cys residues are present in all C. elegans claudins. Interestingly, many of vertebrate claudins have one or two of these motifs or signature. Although most of vertebrate claudins has CLAUDIN3 signature, no such signature was found in the nematode claudins. All of coding sequences of the nematode claudins were isolated from cDNA libraries, therefore they are all expressed in the worm. Some ESTs were reported for CLC-1 and -3, previously. Expression of these claudins was studied with GFP-tagged molecules. All of claudins is expressed in spermatheca, intestine and hypodermis. CLC-1 and -4 were expressed strongly in pharynx, and sometimes localized at cell-cell junctions. They are also seems to be expressed in excretory-secretory system. CLC-1::GFP is also expressed at cell-cell junction of vulva. Localization of CLC-1 is under study with HA-tagged molecule. Preparation of antibodies against the nematode claudins was very difficult so far. But, affinity purified antibodies raised against loop 1 of CLC-1 seem to be useful for CLC-1 detection in the worm. Results with these antibodies were very similar to those obtaind with GFP-tagged CLC-1. To see if these claudins function as mammalian claudins do, i.e. barrier function, penetration of TRITC-dextran (MW=10,000) was checked after injection of dsRNA's (RNAi). Experiments with full length CLC-1 dsRNA showed that barriers for the high molecular weight dye were damaged by RNAi, in other words, penetration of the dye to pharynx and some other tissues were observed. Similar experiments with other claudins did not detect any barrier damage. This is because the dye only goes into entrance of intestine under normal condition, therefore, we tried weak osmotic shock to deliver the dye to entire intestinal lumen and excretory-secretory duct system. Under this condition, control injection of dsGFP did not result in penetration of the dye to other area of the body. On the other hand, RNAi with the combination of CLC-1 and -4 RNA's resulted in penetration of the dye to 72% of the worm from pharynx, intestine and vulva (or from excertory-secretory system) to the body, whereas barrier was damaged only 40% of worm with a combination of CLC-3 and -4. RNAi effects with other combination of CLC's will be reported, and effects of RNAi to the retention of the sperm in spermatheca will be studied. Accordingly, claudins of C. elegans seems to function as barrier at least partly. Other functions, if any, will be surveyed. We would like to thank excellent technical assistance of Miss. Akiko Kamamoto, without her help this project could not be completed.