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

Maruyama IN (1987) Worm Breeder's Gazette "molecular cloning of unc-13."

Maruyama IN

[Worm Breeder's Gazette, 1987]

The unc-13 gene is located 0.025 mu to the right of unc-15 on the centre of LG I. The mutants have a distinctive phenotype; paralysed, kinky and small, and show irregular pharyngeal pumping. More than 30 alleles, including 5 amber mutants, have been isolated and the genetic analysis has been well investigated. It is curious that unc-13(e309) allele is specifically suppressed by sup-6 mutation which is located on LG II, and which does not suppress amber or ochre mutation. In the last gazette, we reported, based on an EM serial reconstruction of a part of ventral cord, that the largest pair of interneurons in the ventral cord of unc-13(e450), AVAL and AVAR, had wrong gap junctions to the excitatory motoneurons of the classes VB and DB, as a major lesion. Towards understanding a molecular mechanism to specify neural connectivity, we have cloned the unc-13 gene as a first step. The strategies employed were as follows: 1) We identified the location of unc-15 gene on 'the genome map' by walking one step both sides of Paramyosin DNA which was isolated by Hiroaki Kagawa. 2) Using Southern hybridization of a set of cosmid clones to a semi- balanced sDf6/unc-15(e73) genomic DNA, the orientation of the contig on the genetic map was determined by locating the sDf6 deletion end point which is located in between unc-15 and unc-13 on the genetic map.

Kagawa H et al. (1990) Worm Breeder's Gazette "CHROMOSOME MAPPING OF mec-1 AND tmy-1, TROPOMYOSIN GENE"

Kagawa H, Coulson AR, Chalfie M, Sugimoto K, Ando H

[Worm Breeder's Gazette, 1990]

To clone the kra-1 gene which might encode a nicotinic receptor associated NMDA receptor-like ion channel (Hideki Ando and Hiroaki Kagawa, in this issue), mec-1 gene was chosen as the starting point for chromosome walking. 11 lambda clones crossreacted with 5 unique fragments from 2 Tc1 induced mec-1 worms were mapped on the chromosome map LGV as was shown in the bottom. One of the clones; HK#9.1 was sensibly located on the position which was calculated by the average relation value between genetic and physical map. We started to RFLP experiment on gamma ray induced mutants to confirm the position of the mec-1 gene by using candidate cosmid clones. International cooperation will accelerate to finish the jig saw puzzle. Tropomyosin gene cloning was nearly finished but still continuing by some unlucky (The worm gazette Vol.10 #3 1988). Any genomic library do not contained tropomyosin gene by some reason of sequence similarity to lambdoid phage. We have already cloned two genomic restriction fragments which contained 6/8 exons. By screening YAC library with one restriction fragment as a probe, 'tmy-1', tropomyosin gene was mapped on the left of unc-54 LGI. The result was independently confirmed with a different approach by Larry Shriefer and Bob Waterston (in this issue). They are doing experiment on searching a candidate mutant of tropomyosin gene defect. Full length cDNA clone was also obtained from new cDNA library (kindly supplied by Barsted and Waterston). Complete cloning business shows the reason why the gene was not packed in a phage particle. [See Figure 1]

Rioux SD et al. (1986) Worm Breeder's Gazette "full length abundant protein and message from unc-15 Tc1 alleles."

Waterston RH, Rioux SD

[Worm Breeder's Gazette, 1986]

We have been analyzing spontaneous unc-15 alleles (r404, r405, r406, r407, r408) isolated from a mixed Bristol/Bergerac strain containing unc-105(n490) by Phil Anderson and Amy Bejsovec (Madison, WI). These unc-15 alleles revert spontaneously to wild type and have better muscle organization and better movement than the null e-1214. We used a lambda clone containing unc-15 specific sequence, kindly provided by Hiroaki Kagawa (Okayama U, Japan), to probe Southerns. Using several different enzymes we observed RFLDs that were 1.6kb larger in r408 than in an isogenic revertant. In order to determine if Tc1 was the cause of the mutation in r408 we cloned and sequenced this region of unc-15. The mutation was indeed due to Tc1 and the insertion site in r408 has homology to the consensus sequence derived by Ikue Mori et al (see this newsletter). [See Figure 1] The 600bp of DNA flanking the insertion site is not AT rich nor does it contain any long 0RFs with the predicted alpha-helix protein structure. unc-15 specific message is present in total and poly-A selected RNA from mixed worm populations in all of these mutants. The unc-15 message is the same size and in roughly the same amount in mutants, isogenic revertants, and N2, and there is no Tc1 hybridizing sequence in the unc-15 message. At the protein level paramyosin is easily detected on SD6 gels of whole worm extracts from these mutants. However, the amount of full length paramyosin detected by polyclonal and monoclonal antibodies in these alleles is reduced several fold from wild type levels. Just how the insertion of Tc1 into the unc-15 region produces these weak alleles is not known. From this preliminary data Tc1 appears to be reducing the amount of paramyosin produced. The DNA sequence flanking the insertion site in r408 is not intron or exon but may be in 5' or 3' regions of the gene. Further analysis of all of the alleles will be necessary to explain these observations.

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.

Sakube Y et al. (1994) Worm Breeder's Gazette "Studies on the Ryanodine Receptor Gene, ryr-1, kra-1 and unc-68 Genes in the LG V."

Ikeda T, Sakube Y, Kagawa H

[Worm Breeder's Gazette, 1994]

Studies on the ryanodine receptor gene, ryr-l, kra-l andunc-68 genes in the LG V Yasuji Sakube, Tatsuji Ikeda and Hiroaki Kagawa Molecular Biology, Department of Biology, Faculty of Science, Okayama University, Okayama Japan 700 c5191 8@jpnkudpc Ryr-l; The worm has the ryanodine receptor gene (vol. 12 #3 p.51, Vol. 13 #2 p58, Sakube etal.) and the functional protein could be the gene product (Vol. 12 #4 p.69 Maryon et a/.). We have completed the structure of the gene on the cosmid clone M04C11 that spans 30 kb and encodes 46 exons. The gene locates between CeLip-1 and odc-lLG V. To know the place of expressed gene, promoter-lyr-1/lacZ fusion plasmids were injected into worms and were analyzed the activity of, beta-galactosidase. At the moment, the ryr-1 gene was expressed in many tissues under the control of 5'UTS. Fusion plasmids having 4.8 k and 2.7 kb of 5'UTS were expressed in muscles. 1.5 kb fragment was expressed in intestine and nerve. Fragments having 1.3 kb and 0.8 kb were expressed in muscles or intestine and nerve cells. At the moment we have not a conclusive result. We also isolated cDNA clones from a library and products of RT PCR and constructed expression plasmids for antibody isolation. kra-l and unc-68; Ando eta/. isolated a mutant kra-l (ketamine response abnormal) and mapped near unc-68 LG V and speculated that a 0.5kb transcript might be a gene product of unc 68 (Vol. 12 #4 p.33 Ando et al.), but the result was over estimate. The recent experiment shows that mutation rescue with a gene fragment recloned from C46C8 was hard to tell the previous speculation. We also constructed a promoter/lacZ fusion plasmid for confirming the expression pattern of the gene fragment of the candidate and obtained this clone specifically expressed in the spermatocyte at L4 to young adult stages as was observed in Spe(sperm specific protein). We have to find another locus at the place of unc-68. We are very much sorry about the previous result. Maryon et al. are isolating a deletion mutant of Iyr-1 by the method of Tc1 excision. We hope that analysis of different approach terminates the conclusion.

Takuwa K et al. (1995) Worm Breeder's Gazette "Tissue Specific Expression of Tropomyosins of Caenorhabditis elegans."

Kagawa H, Takuwa K

[Worm Breeder's Gazette, 1995]

Tissue specific expression of tropomyosins of Caenorhabditis elegans Kyoko Takuwa and Hiroaki Kagawa Department of Biology, Faculty of Science, Okayama University, Okayama 700 JAPAN c5191 8@jpnkudpc.bitnet, hkagawa@sc.okayama-u.ac.jp In C. elegans, there are two different types of muscles; the pharyngeal muscles and body wall muscles. We cloned and sequenced the tropomyosin gene, tmy-1 of the worm which encodes more than three isoforms; two of body muscles and one of pharyngeal muscle. Tmy-1 gene expression was controlled by two promoters and alternative splicing. We have already known the place of the tmy-1 gene expression by injecting series of tmy-1 gene-lacZ fusion plasmids into the oocyte. The expression of promoter activity was detected by histochemical procedure of beta- galactosidase activity. The first promoter controlling two isoforms expressed in the body wall, vulva and male tail muscles. The second promoter specifically expressed in the pharyngeal muscles (Imadzu et al. WBG 13#3p56). To know the results of promoter/lacZ expression were consistent with the results of protein levels, we raised antibody against bacterial produced tropomyosin followed by immunostaining. Fusion protein of b-Gal-CeTMIII was isolated from sonicated bacterial extract by ammonium sulfate precipitation and ion exchange column chromatography. Affinity purification of antibody was prepared by the protocol of Macef and Koch (J. Cell Sci. 92, 61-70 1988), using the proteins immobilized on nitrocellulose membrane. Worms processed with 2X Laemmli buffer run on SDS PAGE. Blotted membrane was stained with 0.01percent Ponseau red in 5percent acetic acid and corresponding band was cut for affinity purificaffon of antibody. Purified protein from SDS-PAGE sometimes is not good antigen for raising antibody for the purpose of histochemistry. We should mention the effect of SDS treatment on the difference between "before" and "after" immunization. Affinity purified antibody by using worm protein was better than that by using bacterial produced protein. Affinity purified anti-CeTMIII antibody stained pharyngeal muscle conversely anti CeTMI/CeTMII antibody stained body wall muscles by indirect immunofluorescence microscopy. Affinity purified antibody also detected a low molecular mass of CeTMII (256 amino acids) on Western blot analysis. These results confirm that promoter-lacZ micro injection experiments were really the result of the transcription and translation of the correct gene. In our micro injection experiments on troponin I (Kuroda et al), troponin C (Matsumoto et al) and ryanodine receptor genes (Sakube et al., WBG 13#2p58), these genes expressed in the body wall muscles. This suggest that muscle contraction in the body wall could initiate with calcium signal of thin filament linked; troponins-tropomyosin. This might suggest that pharyngeal muscle contract with thick filament linked; CaMd-myosin light chain pathway. Upstream carrier of calcium in the pharyngeal may be inositol 1,4,5 triphosphate. Yuji Kohara et al and Baylis et al. isolated the cDNA clones of InsP3 which might express in the pharyngeal (WBG 13#4p18, WBG 13#4p68) .