Worm Breeder's Gazette 9(3): 75
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.
Myosin-3: The myo-3 gene with extensive flanking segments (19kb total; myo-3 DNA sequences were provided by Nick Dibb) was inserted into a vector already carrying sup-7 to yield the plasmid pSAM. This plasmid was injected into tra-3 (e1107am) XX hermaphrodites. Four independent transgenic lines carrying the amber suppressor were selected as described (EMBO J. Oct 86; also see next article). The four resulting loci are designated e2185 to e2188. Homozygous lines have been derived for all four suppressors; e2188 maps on the right arm of X (near unc-9), while e2185 and e2187 both map on chromosome II. e2186 has not been mapped but is unlinked to myo-3.In the last newsletter, RW described a recessive lethal mutation, st378, which behaves as expected for a myo-3 null mutant. The tight linkage (<0.3 between st378 and the marker sma-1 can be used to test for and follow suppression of the st378 mutation . Three of the four transgenic myo-3 185, e2187, and e2188) were found to complement the st378 mutation (i.e. st378 homozygotes are viable in the presence of any of these three loci; n.b. st378 is not suppressed by other amber suppressors [other transgenic amber suppressors or sup- 7]). This indicated that the transgenic myo-3 gene was indeed being expressed in these three lines. Not surprisingly, the myo-3 and sup-7 activities are genetically linked in each case. Two of the transgenic loci (e2187 and e2188) also have sup-3 activity (which is indicative of myo-3 overexpression) in that they suppress null mutations in unc-54 (e190 and e1O92), as well as the unc- 15 missense mutation e73. The phenotypes of different combinations of the myo-3 and unc-54 nulls with each of the suppressors suggests a range in levels of myo-3 expression that can be summarized as follows: e2187 > e2188 > wild type myo-3 > e2185 The tissue specificity of expression was tested using isotype specific monoclonal antibodies to the myosins that were kindly provided by David Miller. A mixture of rhodamine labeled antibody 5-6 ( against myo-3 product) and flourescein labeled antibody 5-8 (against unc-54 product) was used to stain squashed animals. In wild type, both of these antibodies show a high degree of specificly for the body wall muscle. The the tissue specificity in each of the three suppressed st378 homozygote lines was exactly as observed in wild type. In addition each of the three lines showed the alternating filament pattern of the two antibodies which was shown by Miller et al. (Cell 34,477) to result from differential localization of the two different myosins within the muscle thick filament. unc-54: A cosmid (from Alan Coulson and John Sulstons files) which covers the whole of unc-54 coding sequences as well as large 5' and 3' flanking sequences was injected into unc-54 (e190) adults (e190 is a deletion in unc-54.) In addition to a dozen apparent cases of transient expression only in the Fl, three lines with improved movement and restored egg laying were obtained. Each of these three lines has has been maintained for many generations by passaging the well co-ordinated animals. Of the three lines, one behaves just as wild type and two are somewhat more sluggish than wild type. The first (strong) line grows as a homozygote; the unc-54 suppression activity ( designated e2189) has been mapped to chromosome III. No homozygote lines have yet been obtained for the other two (weak) loci e2190 and e2205. Some estimate of e2189 expression can be obtained from its suppression of the unc-54 dominant mutation e1152 -- it appears that the activity of e2189 is within two fold of the wild type unc-54 locus. Tissue specificity of unc-54 expression was investigated using the same mixture of monoclonal antibodies described above. No staining by antibody 5-8 was seen in e190 animals (5-6 staining of these animals looks somewhat disorganized but is still restricted to filaments in body wall type muscle). The patterns of tissue specificity in the the two suppressed lines examined (e2189;e190 and e2190;e190) were identical to those seen in wild type and (as with the myo-3 transformed lines), the alternating filament pattern was still observed (may have been slightly disorganized in e2190;e190). The transgenic suppressors were used to construct doubly transgenic lines, carrying null mutations in both body wall myosin genes along with suppressors for each (e2189+e218) or e2189+e2187). These have the sma-1 marker used to follow st378 but otherwise behave exactly like wild type C. CURRENT ADDRESSES: AF at Carnegie Institute Department of Embryology, 115 West University Parkway Baltimore Md. 21210 Tel: 301-467- 1414 RW at Department of Genetics, Washington University, 660 South Euclid, St. Louis, Mo. 63110 Tel: 314-362- 2722 Although sma-1 was used to follow st378, in all mapping and complementation experiments, the actual presence of st378 was directly confirmed to eliminate rare recombination events in the sma-1 - st378 interval. We used sma-1(e30) derived at an early stage from the original CB30 version of for all of these experiments (i.e. not the mutator version described by C. Trent and C. Link--see J. Hodgkin's note in the last gazette). The original sets of injections were performed with a mixture of two unc-54 cosmids, both of which should cover the gene. Subsequently, one of these has proven sufficient by itself. The total number of animals so far injected with the active cosmid is 49