Worm Breeder's Gazette 12(3): 13 (June 15, 1992)

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.

Selection of Mutants Based on Histochemical Staining Patterns

Guofeng Xie[1], Latha Ravi[1], Stephanie Jones[1], Yiwen Jia[1], Joseph Culottit[2], Michel Hamlin[2], Eric Aamodt[2]

[1]Louisiana State University Medical Center, P.O. Box 33932, Shreveport, LA 71130-3932
[2]The Samuel S. Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5 G1XS; Merck Sharp & Dohme, Rahway, NJ 07065

We have used the histochemical staining method described in the accompanying article (G. Xie and E. Aamodt) to screen for mutations that alter the pattern of either carboxylesterase activity in wild-type strain N2 or ,B-galactosidase activity in a strain (NW640) that contains a mec-7 lacZ fusion gene.

Wild-type adult C. elegans stain for esterase activity in their gut and in three clusters of cells (neurons?) just behind the nerve ring. We have isolated four mutant strains following EMS mutagenesis of N2 that are also stained for esterase in (as yet unidentified) cells in their heads and in their dorsal and ventral nerve cords (the particular cells stained varies with the mutation). We isolated two other mutant strains that are stained in their pharynx muscle cells. This phenotype is similar to the pharynx expression of the major C. elegans esterase gene ( ges-1 )that has had all but 1220 bp of upstream noncoding sequence removed (see Aamodt, Chung and McGhee (1991) Science 252:579) and the esterase staining patterns of wild nematode species (see the accompanying article by G. Xie, L. Ravi and E. Aamodt on esterase expression patterns in wild nematode species). While the ges-1 gene product is the only esterase present in embryos, adults also express several minor esterase species that can be detected on isoelectric focusing gels (McGhee and Cottrell (1986) Mol. Gen. Genet. 202:30). Until we cross these mutations into a ges-1 (0) background, we will not know whether these mutations are affecting the expression of ges-1 or one of the other esterase genes. These mutants have been back-crossed to wild-type once but we do not know yet where they map or whether the mutations are cis or trans to the esterase genes.

We have also begun to screen for mutations that alter the expression of a mec-7 lacZ fusion gene constructed by Michel Hamlin and Joe Culotti. This strain has been transformed with plasmid pNW115 which is made with Andy Fire's vector pPD16 .51and contains 852 bp upstream of the mec-7 start codon, 792 bp of mec-7 coding region, the lacZ coding region in frame with mec-7 (the nuclear localization signal was removed), the last 47 bp of the unc-54 coding region and 1276 bp downstream of unc-54 .This construct was transformed into N2 along with Jim Kramer's pRF4 plasmid that contains the dominant mutant rol-6 gene. The extrachromosomal array formed in this way was then linked to chromosome I by the Kari et al. irradiation procedure (WBG 11[3]14) . We are screening EMS mutagenized NW640 for mutations that alter the pattern of expression of the fusion gene. We appear to be isolating mutants at a reasonably high frequency. In the two mutant strains that we have begun to characterize there is greatly increased ,ß-galactosidase expression in the touch neurons, the FLP, PVD and ALN neurons and expression in other nerve cells (as yet unidentified) where the enzyme was not previously detected. We do not know whether the cells that are stained only in the mutants represent new expression in cells where the gene was not previously expressed or simply expression that is now detectable due to the enhanced expression of the gene.

Literature Cited:

G. Xie , and E. Aamodt. (1992). WBG 12.3.12

Aamodt, Chung and McGhee (1991) Science 252:579

G. Xie, L. Ravi, and E. Aamodt. (1992). WBG 12.3.11a

McGhee and Cottrell (1986) Mol. Gen. Genet. 202:30