Yohann Duverger et al. (2007) International Worm Meeting "A semi-automated high-throughput approach to the generation of transposon insertion mutants."

Yohann Duverger, Jonathan Ewbank, Christophe Beclin, Sarah Scaglione, Dominique Brandli, Jerome Belougne

[International Worm Meeting, 2007]

To understand gene function, there is a clear need for the generation of as many stable mutant lines in as possible. Two KO projects are currently underway in North America (http://celeganskoconsortium.omrf.org/) and Japan (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and have generated thousands of mutants. To these can be added the null alleles for several hundred genes produced using other PCR-based technologies or generated in classical genetic screens and available from the CGC. An alternative approach to the generation of mutants is via the use of transposons. Recently, a method using the mariner-like element Mos1 from Drosophila has been established (1). It was used in a pilot-scale project to generate random insertions throughout the genome (2). Following on from this manually project, as part of a collaborative effort funded by the European community (Nemagenetag; see abstract by Robert et al.), the French National Genopole network and support from Union Biometrica, we have developed a semi-automated high-throughput method for mutant production and screening. Using the UBI COPAS Biosort and a robotic system for PCR and electrophoretic analysis (using a 400-well gel format), we have developed a capacity to handle several thousand nematode strains in parallel for multiple generations (3) and have already generated more than 45,000 individual strains carrying Mos1 insertions. We will the describe method in detail, report recent improvements that have pushed our production capacity to above 5,000 mutant strains/month and explain how the method can be easily adapted to forward and reverse genetic screens. 1. Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. & Jorgensen, E. M. (2001) Nature 413, 70-4. 2. Granger, L., Martin, E. & Segalat, L. (2004) Nucleic Acids Res 32, e117. 3. Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. & Ewbank, J. J. (2007) Nucleic Acids Res 35, e11.

Robert, Valerie J P et al. (2009) International Worm Meeting "MosLIB: A PCR-screenable Mos1 insertion library."

Robert, Valerie J P, Bessereau, Jean-Louis

[International Worm Meeting, 2009]

MosTIC (for Mos1 excision transgene-instructed gene conversion)1 provides a means to engineer the Caenorhabditis elegans genome. In MosTIC, transgene-instructed gene conversion occurs during the repair of a DNA double-strand break generated by the excision of a pre-existing Mos1 insertion located in the genomic region to engineer. Mos1 is a DNA transposon isolated in Drosophila mauritiana. In C. elegans, expression of the Mos transposase is sufficient to induce the mobilization of Mos1 from an extrachromosomal array into the genome2. Such genomic insertions are indispensable reagents to initiate MosTIC. Here, we present the development and the characterization of MosLIB, a Mos1 insertion library consisting of 40,000 independent clonal strains containing Mos1 insertions3. MosLIB is made of 719 pools of 48 to 72 clonal strains. Each pool is represented by a duplicated frozen nematode stock and the corresponding genomic DNA. We estimated that at least 80,000 Mos1 insertions are present in MosLIB, 65 percent of them being close enough to coding regions to be valuable reagents to engineer genes by MosTIC. To screen MosLIB, we first perform a single PCR on the DNA stock with one primer in Mos1 and one specific primer designed in the genomic region of interest. Once a positive signal is obtained and its identity confirmed by sequencing, we thaw one tube of the corresponding nematode stock and we make 20 to 40 pools of 20 animals which survived freezing. These pools are screened by PCR for the presence of animals carrying the insertion of interest and rounds of sib-selection are made until a single homozygous animal carrying the insertion of interest is isolated. To optimize the screening of MosLIB, we now aim at using high-throughtput sequencing technologies to map the Mos1 insertions present in MosLIB. We would like to thank Y. Duverger, J. Ewbank and members of the Bessereau''s lab for their help in the making of the library. 1.Robert, V. and Bessereau, J. L. Embo J 26, 170-83 (2007). 2.Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. and Jorgensen, E. M. Nature 413, 70-4 (2001). 3.Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. and Ewbank, J. J. Nucleic Acids Res 35, e11 (2007).

Stefano Canevascini et al. (2006) European Worm Meeting "Protein O-fucosylation is Important for Normal Distal Tip Cell Migration"

Ruth Chiquet-Ehrismann, Jasmin Althaus, Dominique Klein, Marianne Grob, Krisztina Kozma, Jan Hofsteenge, Stefano Canevascini

[European Worm Meeting, 2006]

Stefano Canevascini, Krisztina Kozma, Marianne Grob, Jasmin Althaus, Dominique Klein, Ruth Chiquet-Ehrismann and Jan Hofsteenge. Protein glycosylation is crucial for the development and growth, as well as the normal functioning and survival of an organism. However, in most instances we do not know the exact function and the mechanism of action of glycan modifications at the molecular level. We want to elucidate the function of glycosylations that occur on thrombospondin type 1 repeats (TSRs). TSRs are cysteine- and arginine-rich modules of about 60 amino residues in length that are part of many proteins with important physiological functions, like e.g. inhibition of angiogenesis, cell- and axonal guidance and regulation of complement in higher organisms. TSRs undergo two different types of glycosylation: i) C-mannosylation of tryptophan residues and (ii) the addition of O-linked fucose to serine and threonine residues, followed by extension with a single glucose to form Glc-beta1,3-Fuc-O-Ser/Thr. We have begun to study the latter process in C. elegans, in order to reveal its possible function. The C. elegans genome encodes 29 TSR-containing proteins, some of which have been found to be important for normal development, like UNC-5, GON-1, ADT-1 and NAS-36.. We have demonstrated that C. elegans pad-2 encodes the fucosyltransferase (POFUT-2) that modifies TSRs. Extracts from HEK293T cells overexpressing PAD-2 show a 10-fold increase in transferase activity towards a TSR. Furthermore, product analysis by mass spectrometry of isolated peptides showed the fucose to be linked to the correct Thr residue. In contrast, no increase in TSR fucosylation was observed by overexpressing C. elegans POFUT1, the enzyme that fucosylates EGF-like repeats (encoded by C15C7.7). We are characterizing the pad-2(tm1756) mutant that we received from the NBP, Japan. The tm1756 strain is homozygous viable and lacks all fucosyltransferase activity towards a TSR. The mutant does not show embryonic lethality nor morphogenetic defects (Menzel et al., Genomics 84: 320, 2004), but a precocious dorsal migration of the anterior gonad arm is observed. The latter is reminiscent of the phenotype resulting from the precocious expression of UNC-5 in DTCs (Su et al., Dev. 127: 585, 2000). Genetic experiments show that unc-6, unc-5 and unc-40 genes are epistatic to pad-2, suggesting that pad-2 is acting as an inhibitor of the netrin pathway in DTCs. We are now testing whether the TSRs of C. elegans UNC-5 are fucosylated in vivo and whether this modification is necessary for the pad-2-mediated inhibition of the DTC ventro-dorsal migration. Preliminary evidence indicates that C. elegans also contains the glucosyltransferase activity that attaches glucose to the fucose It will be of interest to examine its possible function.

Wood WB et al. (1994) Worm Breeder's Gazette "Low Temperature Affects Embryonic Handedness Reversal."

Bergmann DC, Florance A, Wood WB

[Worm Breeder's Gazette, 1994]

LOW TEMPERATURE AFFECTS EMBRYONIC HANDEDNESS REVERSAL Bill Wood, Arny Florance and Dominique Bergmann Department of MCD Biology, University of Colorado, Boulder C. elegans embryos become left-right asymmetric at the 6-cell stage witlf a handedness which is essentially invariant among N2 animals reared at 20, and which persists into adulthood, determining the asymmetric placement of the gonad, intestine, the coelomocytes, and several other cells. Reversal of embryonic handedness at the 6-cell stage by micromanipulation results in development of healthy adults with all left-right asymmetries reversed. l Spontaneous reversal occurs at a frequency of a few percent among animals developing embryos treated with chitinase at the 2-cell stage to remove the egg shell.2 We have now found that when N2 are reared at 10, the frequency of animals with reversed gonad handedness as adults increases to above 0.5%. Those examined by Nomarski microscopy exhibited handedness reversal of the ventral nerve cord (cell bodies on the left) and reversed placement of the coelomocytes as well as the intestine and gonad, suggesting that these animals developed from reversed embryos. To determine the cold-sensitive period for handedness reversal, young gravid adults reared at 20 were placed at 10 for 2 hours, then transferred to new 20 plates and subsequently transferred to fresh 20 plates at 2-hr intervals. Eggs laid during the cold pulse (interval l; exposed to 10 after fertilization), the first 2 hr after return to 20 (interval 2; exposed to 10 either just before or just after fertilization), and each of the four subsequent 2-hr intervals at 20 (3-6, exposed to 10 before fertilization) were allowed to hatch and develop at 20 into adults, which were then scored for gonad handedness. Among animals developing from embryos exposed to 10 after fertilization (interval 1), no reversals were' observed (N=1,956), whereas among animals developing from oocytes and sperm exposed to 10 before fertilization (intervals 3-6), the frequency of reversal was 0.21% + 0.09% (N=9,223). Surprisingly, although left-right asymmetry first appears at the 6-cell stage, and the embryo's left and right are highly unlikely to be specified until the 4-cell stage, I the cold-sensitive period for reversal is before fertilization. This result suggests that the effect is on the gametes. We can imagine two possible explanations: 1) Production of some eggshell precursor component, synthesized during oogenesis, could be cold sensitive, such that embryos with "soft" eggshells are produced at 10, leading to a low frequency of reversal as seen in chitinased embryos.2 2) Some handed structure in a gamete, such as the sperm centriole for example, may normally dictate the skewing of the ABa and ABp spindles that leads to handed asymmetry in the embryo;l when this structure is exposed to 10 its subsequent ability to dictate handedness could be impaired, leading to a low frequency of reversal. We are currently carrying out experiments with chilled males to determine whether egg or sperm is the cold-sensitive gamete, as well as examining effects of other treatments on handedness reversal. Finally, we are continuing small scale screens for maternal-effect mutations that result in a high frequency of embryonic reversal. None have been found, but so far we have screened only about 1000 EMS-mutagenized genomes. I Wood, W. B. (1991) Nature 349: 536-538. 2 Wood, W. B. and Kershaw, D. (1991) In Biological Asymmetry and Handedness, CIBA Foundation Symposium 162: 143-164.