Worm Breeder's Gazette 12(5): 59 (February 1, 1993)

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.

Killer on the Loose: Ectopic Expression of mec-4 (d)

Maud Kinnell[1], Si Qun Xu[2], Harbinder Singh[3], Andy Fire[2], Monica Driscoll[3]

[1]Department of Molecular Biology and Biochemistry, Rutgers
University, Center for Advanced Biotechnology and Medicine, 679
Hoes Lane, Piscataway, N.J. 08855

[2]Carnegie Institution of Washington, Department of Embryology, 155 W. University Parkway, Baltimore, MD 21210
[3]Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, N.J. 08855

mec-4 mutations that encode substitutions of large side chain amino acids for a conserved Ala (position 442 in the truncated mec-4 cDNA clone) induce swelling and degeneration of the touch receptor neurons, the cells in which mec-4 is normally expressed. We have begun testing the effects of ectopic expression of mec-4 (d)by fusing mec-4 cassettes to various C. elegans promoters.

The Genomic mec-4 cassette.

Our deletion analysis of the mec-4 gene established that removal of the first 1066 nucleotides from the 5' end of our published genomic sequence abolishes function of the gene in complementation assays. This deletion removes all S' regulatory sequences and the beginning of the putative coding region such that the first 19 amino acids of Mec 4 would be missing if the protein was translated.

Expression of mec-4 alleles from the mec-7 promoter. We first used

the mec-7 promoter to demonstrate that genomic mec-4 sequences

after nucleotide 1066 can function as a mec-4 cassette. The mec-7

gene is expressed at high levels in the touch cells and at lower

levels in the PVD cells, the ELP cells, the BDU cells, a pair of cells

in the ventral ganglion and a few cells in the tail (Hamelin et al.,

EMBO J 11:2885, 1992; M. Chalfie and colleagues, pers. comm.).

Placing the mec-7 promoter in front of mec-4 ^1066restores mec-4

expression of the mec-7 mec-4 (+)fusion gene complements mec-4 (r)

mutations. Note that this implies that the first 19 amino acids of Mec-4 are dispensable for function of this protein in the touch cells. A mec-4 (d)cassette fused to the mec-7 promoter induces death of the touch receptor neurons and the other cells in which mec-7 is expressed. The observed ectopic killing of some of these cells was in part expressed from results of studies of double mutants of mec-4 (d)and egl-44 , egl-46 and sem-4 (M Chalfie and colleagues, pers. comm.)

Expression of mec-4 (d) from a heat shock promoter.

We have fused the mec-4 (d)cassette to the hsp16 /2promoter and examined animals transformed with this gene for degenerations induced after heat shock Animals observed after four hours of recovery from four hours of heat shock at 34° exhibited many dying cells throughout their bodies. Eggs also exhibit many swollen cells. Because transformants are mosaic for the presence of the fusion gene it is not clear what cells, if any, are refractory to the deleterious effects of mec-4 (d).We have constructed several integrated lines that we can now use to study the types of cells that suffer degenerative death. These lines are fairly sickly even when reared at low temperature.

Expression of mec-4 (d) from the unc-54 promoter.

In order to examine the consequences of expression of mec-4 (d)in muscle cells, we have fused the gene to the unc-54 promoter. In control experiments with a unc-54 p- mec-4 /lacZfusion, we observe ß-galactosidase in muscle but not in touch cells. In lines expressing mec-4 (d)from the unc-54 promoter we observe a range of phenotypes: animals that roll as larvae often become nonrollers ( rol-6 ( su1006 )is coinjected in these experiments), some animals appear hypercontracted and some animals bag. A clearlike phenotype is often observed--the gut remains prominent, but the gonad and muscles appear transparent (if they are there at all). At higher magnifications we can see many small vacuoles at positions consistent with body wall muscle. Thus, it appears that muscles expressing mec4 (d)contain multiple small vacuoles rather than the single large swelling exhibited by degenerating neurons. We are using antibody staining to clarify what happens to the muscle.

We conclude that ectopic mec-4 (d)expression can have drastic consequences for multiple cell types. mec4 (d)may prove generally useful for eliminating the function of cells for which regulated cell-specific promoters have been characterized.