Worm Breeder's Gazette 10(1): 60

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.

Control of unc-54 Expression

A. Fire and S. Harrison

Figure 1

As reported in a previous gazette article (A. F.  and R.  Waterston 
WBG #9.3), cosmids carrying the unc-54 gene contain all of the cis-
acting information necessary for proper expression of the gene.  We 
are attempting to define the sequence elements mediating body wall 
specific gene expression.  To do so, we constructed plasmids carrying 
unc-54 sequence which have been deleted and mutated in vitro.  These 
plasmids are injected into oocytes of animals carrying the mutation 
unc-54(e190).  e190 is a deletion eliminating unc-54 product [the 
major myosin heavy chain protein] and greatly diminishing unc-54 
message levels.  This allows both transient and stable expression 
assays for the injected DNA.
Transient Expression:  After injection of functional unc-54 plasmids,
a high fraction (5%-25%) of the injected oocytes give rise to rescued 
progeny, as assayed both by improved movement and egg laying.
Stable Transformation: Most of the transiently rescued animals do 
not breed true; that is all progeny are unc.  In a small fraction of 
cases the rescued animal has some rescued progeny and a transformed 
line is established.  Frequencies of stable transformation vary 
greatly (3-20% of injected animals), with the factors influencing 
transformation frequency still not understood.  Stably transformed 
lines are grown up and analyzed for the presence of DNA and RNA from 
the initial plasmid, and stained with monoclonal antibodies (from 
David Miller) to localize unc-54 protein expression.  An anti myo-3 
monoclonal with a different fluorescent tag is used as internal 
control and marker; unc-54 and myo-3 proteins localize to the same 
muscle cells in wild type animals.  A considerable variability in the 
level of expression from the transgenic loci constructed with the same 
input DNA is observed.  The level of expression does not correlate 
with the copy number of the injected DNA and is thus likely to reflect 
modest position or contextual effects.  Copy numbers in the lines vary 
from 0.5-6 per haploid genome.  A single exceptional line with about 
60 copies of plasmid pUNR-54 (used as a starting point for the 
deletions) actually expresses very poorly.
The unc-54 gene does not require extensive flanking sequences for 
proper expression.  On the 3' side, deletions which leave 1000nt 
beyond the poly A site exhibit normal expression.  More surprisingly, 
almost no 5' sequence seems to be required for expression.  The 
startpoint of transcription has been mapped to 80-85 nt upstream of 
the AUG (Originally done by Mike Krause and repeated by us.) Deletions 
which eliminate some or all of the upstream region still function in 
both transient expression and stable transformation, with the wild 
type pattern of expression observed in all of the stably transformed 
lines.  More surprisingly, a deletion which eliminates the entire 5' 
flanking region and 50 nt of the transcribed leader sequence is also 
functional in both transient expression and stable transformation.  A 
transformed line carrying this deletion expresses unc-54 myosin in 
exactly the correct subset of cells.  Reassuringly, a deletion which 
removes the AUG and first four codons is nonfunctional for expression.
Primer extension analysis of RNA from strains rescued with 550 or 
130 nt of DNA upstream from the transcriptional start show the wild 
type pattern, suggesting that 130 nt of 5' flanking DNA is sufficient 
for proper initiation of transcription as well as proper regulation.  
RNA from the line transformed with the +50 deletion seems to start at 
a set of sites just adjacent to the gene within plasmid sequences.  
This suggests that a tissue specific regulatory element (an enhancer?) 
within the transcribed region can stimulate transcription from a 
variety of upstream start sites.
Expression of unc-54:  -galactosidase fusion 
Three different unc-54:  -gal fusion proteins have been integrated 
into the genome of tra-3(e1107am) animals using sup-7 as a selectable 
marker.  These fusions were constructed conservatively so that both 
the 5' and 3' ends of the resulting chimeric gene derive from unc-54.  
Each of these proteins is expressed at high levels in transformed 
lines.  In each case (four lines total), the expression is limited to 
the body wall type muscle cells.  In the first two cases the staining 
is throughout the cells, with distinct blue staining outlining the 
four muscle quadrants.  In the third case the staining is primarily 
nuclear with each of the body wall type muscle nuclei staining dark 
blue.  Expression of these fusions is much higher (10-100 fold) than 
the expression of the HSP:  -gal and myo-2:  -gal fusions that has 
been reported previously.  Whether this results from differences in 
transcription due to the unc-54 'promoter' or to differences in 
message stability due to 5' and 3' unc-54 RNA sequences is not yet 
known.  The ability of the SV40 nuclear localization signal to 
function in construct III but not construct I presumably reflects 
different contexts of that region within the two proteins upon folding.

Taken together, the whole gene expression data and the  -gal fusion 
data suggest that a determinant for tissue specificity is located in 
one of two regions (+50 to +1960 from the start of transcription or -
240 to +970 from the polyadenylation site).

Figure 1