Kanako Hirano et al. (2001) International C. elegans Meeting "GENES SHOWING ALTERED EXPRESSION ACCOMPANIED BY HABITUATION"

Ryuji Hosono, Kanako Hirano

[International C. elegans Meeting, 2001]

Habituation to mechanical tap stimuli in C.elegans belongs to short term memory. For understanding the habituation, it is useful to identify the neural circuit and genes responsible for the stimulation. Wicks et al., determined the neural circuit for tap-mediated stimulation consisting of mechanosensory neurons, interneurons, and motorneurons (1) . However, the exact neuron contributing to habituation was not identified. We therefore addressed this issue by systematically ablating each neuron constituting the network for the mechanical stimuli. We found that the AVDL and AVDR interneurons play a critical role in the habituation in intact animal. And the AVM and ALMLR mechanosensry neurons are important for habituation of the AVDLR ablated animals (2) . For further investigation of the habituation mechanism, it is essential to identify genes contributing to the behavior. In order to identify genes that are differentially expressed as a consequence of repeated tap stimulations, we used the differential display technique to compare mRNA expression patterns. DNA bands of interest, those with signals showing the difference in intensity in control lanes relative to the test lanes, were eluted from the gel, re-amplified, ligated into a plasmid vector and their double stranded sequences were determined. Twenty up-regulated and 30 down-regulated clones were identified. Each DNA band on the DD gel plates was isolated, cloned and its DNA sequence was determined. We found that they are functionally classified into four groups : metabolism, neural function,signal transduction and transcription. Of these following four clones were especially interested. Of up regulated genes, two clones encoding MAPKKK, and GTP-binding protein, and of down regulated two clones encoding transcription factor, and ionotropic grutamate receptor GLR-6 were noticed. As a current work we are studying changes in the expression pattern during the tap stimulation. And further we are investigating cells expressing these genes by the in situ hybridization. 1. Wicks, SR., Rokrig CH., Rankin, C.H., (1996) A dynamic network stimulation of the nematode tap withdrawal circuit : Predictions concerning synaptic function using behavioral criteria. J. Neurosci. 16, 4017 - 4031. 2. Kitamura, K., Amano, S., Hosono, R. (2001) Conrtibition of neurons to habituation to mechanical stimulation in C.elegans . J. Neurobiol., 46, 29 - 40.

Toshihiro Sassa et al. (2001) International C. elegans Meeting "THE C.ELEGANS PROTEIN PHOSPHATASE 1 HOMOLOGUE(CEPP1) IS ESSENTIAL FOR EMBRYOGENESIS AND POST-EMBRYONIC DEVELOPMENT"

Ryuji Hosono, Keiko Gengyo-Ando, Shohei MItani, Shin-ichi Harada, Keiichiro Kitamura, Kanako Hirano, Toshihiro Sassa, Hiroko Ueda

[International C. elegans Meeting, 2001]

Protein dephosphorylation functions as modulator in many biological events. To elucidate the regulatory aspects of protein dephosphorylation, we identified and cloned the gene encoding protein phosphatase 1 homologue( Ce PP1), which was found within cosmid F56C9 located LGIII. From the expression pattern of the Ce PP1::GFP fusion protein and in situ hybridization, the gene is ubiquitously but especially strongly expressed in gonad, neuron, muscle and intestine. Microinjection of double stranded RNA of this gene induced an embryonic arrest at multicellular stages. To characterize in more detail the function of gene( pp1-1 ) encoding Ce PP1, we isolated a mutant( tm291 ) by TMP/UV method, which had a deletion of exon VII of pp1-1 . Hermaphrodites heterozygous for the pp1-1 mutation produce homozygous embryos which can hatch and grow to adulthood. However, progeny produced from hermaphrodites homozygous for its mutation almost (96%) die at multicellular stages during embryogenesis. DAPI staining of embryos revealed that the presence of chromatin bridge and multinucleated cells, suggesting the gene being implicated in chromosome condensation and segregation. However, the staining of the mutant with antisera against alpha-tubulin was normal. These observations are reminiscent of the air-1 /aurora-related kinase gene defective mutants(Schumacher et al ., Dev. 125 4391, 1998) or nuclear lamin( lmn-1 ) defective embryos(RNAi)(Liu et al ., Mol.Biol.Cell 11 3937, 2000). The defect of karyokinesis in the pp1-1 mutants may be elucidated by the following results; Ce PP1 expressed in HEK 293 cells catalyzes Pi release from phospho-histone H3 phosphorylated by PKA. The morphological abnormality of homozygous mutant at the post-embryonic stage is also observed including vulva and gonad formation. These phenotypes are also reminiscent of stu-7/air-2 defective mutants (Woollard and Hodgkin, Mech. Dev., 82 95, 1999). The pp1-1 gene was strongly expressed in the AFD and ASE amphidal neurons. We therefore are ongoing to elucidate the implication of the gene in both neurons. As mutant showed normal movement, we can examine chemotactic and thermotactic behavior which were sensed by ASE and AFD, respectively. Preliminary studies showed that the mutants displayed normal attraction to NaCl, but abnormal thermotactic behavior.

Albrecht MR et al. (1998) West Coast Worm Meeting "dpy-28: AN ESSENTIAL DOSAGE COMPENSATION GENE ALSO REQUIRED FOR MEIOTIC CHROMOSOME SEGREGATION ENCODES A CHROMOSOME CONDENSATION HOMOLOG"

Albrecht MR, Meyer BJ

[West Coast Worm Meeting, 1998]

Dosage compensation is a chromosome-wide process that equalizes X-chromosome gene expression between the sexes, despite their two-fold difference in X dose. In the nematode C. elegans, dosage compensation occurs by halving the level of transcripts from both X chromosomes of hermaphrodites. The genes that are required to establish proper dosage compensation can be split into two groups, those that coordinately regulate dosage compensation and sex determination and those that only regulate dosage compensation. dpy-21, dpy-26, dpy-27 and dpy-28 are in the latter category and are only required for dosage compensation. Cytological and biochemical analysis of the DPY-27 and DPY-26 proteins revealed that these proteins are part of a dosage compensation complex that reduces X-linked gene expression through its sex-specific association with both X chromosomes of hermaphrodites. A reverse genetic approach lead to the characterization of a third component of the dosage compensation complex, MIX-1. The characterization of the cloned dosage compensation proteins suggested that the process of dosage compensation is mechanistically related to chromosome segregation. First, MIX-1 plays essential roles in both dosage compensation and mitosis. Second, the characterized components of the dosage compensation complex share some similarity with the components of the 13S condensin complex, a five protein complex required in vitro for mitotic chromosome condensation in Xenopus. Both complexes contain SMC proteins: MIX-1 and DPY-27 in C. elegans, and XCAP-E and XCAP-C in Xenopus. The SMC family of proteins are conserved from bacteria to humans and are required for various aspects of chromosome dynamics. Furthermore, the non-SMC proteins DPY-26 and the condensin XCAP-H share some similarity, although this similarity is limited to two short motifs. Finally, analysis of dpy-26 and dpy-28 mutants demonstrates that both genes play a role in chromosome segregation during meiosis. We sought to clone the dpy-28 gene to further our understanding of the process of C. elegans dosage compensation and its relationship to chromosome segregation. We mapped dpy-28 using RFLPs to the area between cosmids K11D9 and K01G5 on LG III. This region contains a single gap between two cosmid islands, and several attempts to positionally clone dpy-28 failed. To circumvent the difficulties associated with the positional cloning of genes in areas of the genome with poor cosmid representation, we designed an RNA interference (RNAi) assay to detect RNAs that caused defects in dosage compensation. Using partial sequence data from the YAC Y39A1, which covers the region between K11D9 and K01G5, we identified expressed sequence tags (ESTs) of genes in this region. Cataloging the ESTs resulted in 33 cDNA families. We screened RNAs using RNAi from all 33 cDNA families and identified one single clone that resulted in dosage-compensation-specific phenotypes. We sequenced the potential dpy-28 gene and found that it encodes a predicted protein of 1455 amino acids (166 kD), a size consistent with an unidentified band in the dosage compensation complex. We sequenced the genomic DNA of the mutant allele s939 and discovered that it caused an in-frame deletion of 66 amino acids, confirming the cloning of dpy-28. Sequence analysis of the dpy-28 gene reveals that it is a member of a protein family conserved from yeast to humans, including the condensin XCAP-D2 (T. Hirano, personal communication). The discovery that the non-SMC proteins dpy-28 and XCAP-D2 are shared between the dosage compensation and condensin complexes strongly suggests a common mechanism for establishing chromosome structure and fine-tuning X expression. These findings further support the notion that dosage compensation evolved by recruiting proteins involved in chromosome segregation for the new purpose of gene expression. Because DPY-28 is similar to XCAP-D2, we speculate that DPY-28 will be part of the dosage compensation complex currently consisting of DPY-26, DPY-27 and MIX-1. Antibodies have been generated against DPY-28 in order to test this possibility.