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.

Reintroduced Myosin Genes are Correctly Expressed

A. Fire and R. Waterston

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 > 
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 
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-
RW at Department of Genetics, Washington University, 660 South 
Euclid, St. Louis, Mo. 63110 Tel: 314-362-
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