Worm Breeder's Gazette 13(3): 87 (June 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

hch-1 Rescued

Ryuichi Hishida[1], Takeshi Ishihara[2], Kazunori Kondo[3], Isao Katsura[2]

[1]DNA Research Center, National lnstitute of Genetics, Mishima, Shizuoka-ken 411, Japan
Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan

[2]DNA Research Center, National lnstitute of Genetics, Mishima, Shizuoka-ken 411, Japan
[3]Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192, Japan

Mutations in the gene hch-1 cause defects in both hatching and cell migration (E. Hedgecock et al., Development 100, 365-382, 1987). First, hch-1 mutants exhibit delayed hatching, because the mutant embryos cannot digest proteins in their own eggshell. Second, the descendants of the QL neuroblast of hch-1 mutants fail to migrate posteriorly, but migrate anteriorly, instead. How does hch-1 gene function in these quite different systems?

We have isolated a new allele of hch-1 , ut110 ,from a mut-6 strain in the course of screening larval lethal mutations that cause gross morphological abnormalities (I. Katsura et al., WBG 12-2 pl08 ).Recently we cloned a unique Tc1 -containingfragment (HindIII 3.0kbp) from the genomic DNA of hch-1 ( ut110 ).A YAC filter and some cosmid clones were analyzed with a DNA fragment flanking the Tc1 as a probe. This probe hybridized to YAC clones Y42D5 , Y15F6 , Y52C11 , Y43C6 and cosmid clones C42E7 , F40E10 , M86 , K11E3 , T15A10 , F23C1 .The result is consistent with genetic map position of hch-1 gene (near unc-3 X). Since the Tc1 insertion site is located near either end of the cosmid clones, we did not try to rescue hch-1 worms with the cosmids. Instead, we cloned two DNA fragments containing the Tc1 insertion site, a 15kbp PstI-ClaI fragment and a 9kbp SacI fragment, from the N2 genomic DNA. These clones together with pRF4 as a dominant Rol marker were injected into hch-1 ( ut110 )mutants, and the extrachromosomally transformed worms were irradiated with gamma ray to obtain integrative transformants. The integrative transformants containing the PstI-ClaI fragment or the SacI fragment show no delay of hatching. We are presently trying to rescue with shorter DNA fragments.

Total RNA isolated from mixed staged N2 worms was analyzed by Northern blot using the SacI fragment as a probe. This experiment revealed two transcripts (roughly 3kb and 0.5kb in length) in this region. We plan to determine if these RNAs are encoded by hch-1 gene and to isolate hch-1 cDNA.