Worm Breeder's Gazette 10(2): 87

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.

Requirements for Expression of the unc-54 Gene

Andrew Fire and Susan Harrison

Figure 1

In the last gazette, we reported the beginnings of a deletion 
analysis of the unc-54 gene.  We have since extended this analysis.  
To date, several surprising results have emerged.
Methods: Plasmid clones carrying modified or deleted copies of the 
unc-54 gene are constructed.  These plasmids are injected into the 
oocytes of unc-54(e190delta) animals.  If the particular plasmid is 
active, then a high fraction (5-25%) of the injected oocytes give rise 
to animals that are rescued in the sense that their movement is 
greatly improved over e190 or mock-injected control animals (also the 
rescued animals are often egl+).  A small fraction (3-20%) of the 
rescued animals give rise to heritable transformed lines.  In order to 
assess tissue specificity, these lines are stained with a mixture of D.
Miller's monoclonal antibodies.
Upstream Sequence Requirements: Essentially there are no sequences 
upstream of the unc-54 gene that are required for tissue specific 
expression.  This result is described in some detail in our article in 
the last gazette.  Since then the 'ultimate' upstream deletion has 
been engineered, missing all upstream sequences to +79 from the site 
of transcriptional initiation (6bp upstream of the AUG).  This 
construct is completely active for unc-54 function.  Transformed lines 
carrying the +79 deletion give the wild type pattern of tissue 
specific expression.  Deletion of the next 21 bases (including the AUG)
results in an inactive gene.
Since each of the deletion mutants assayed so far had substituted 
plasmid sequences for the deleted sequences at the 5' end of the gene, 
we decided to replace the 5' flanking sequences with DNA flanking 
another nematode gene.  We chose the myo-2 gene, which encodes a 
myosin isoform made only in pharyngeal muscle (unc-54 and myo-3 are 
expressed in body wall type muscles).  Therefore we substituted a 
region of 1660 bp from upstream of the myo-2 gene for the sequences 
upstream of the unc-54 message (the fusion site was at +50 in the unc-
54 message).  This construct is active in rescuing the paralyzed 
phenotype of unc-54(e190).  This result further supports the 
hypothesis that sequences inside of the unc-54 gene are sufficient for 
its expression in body wall muscle.
When a transformed line carrying the myo2:unc-54 fusion was stained 
with the antibody mixture described above, it was found that unc-54 
protein (but not myo-3 protein) was present in the pharyngeal muscles 
as well as body wall muscles of the transformed animals.  This result 
shows that the observed differential localization of the myosin 
isoforms to the different muscle types is not a result of protein 
stability or differential detection.  Interestingly, the unc-54 myosin 
seems organized into radial filaments when present in pharyngeal 
Intron Requirements: Intron sequences near the start of the message 
are apparently required for efficient expression of the gene.  
Deletion of the first four introns (using a synthetic cDNA clone 
provided by J.  Karn) results in a plasmid (pUNK54delta1234) with 
almost no activity in transformation assays.  By performing a very 
large number of injections few partially rescued animals have been 
obtained with this plasmid, and one transformed line has been obtained.
This line is very sick (e.g.  usually sterile), exhibiting a variety 
of abnormalities.
A variety of plasmids with deletions of different subsets of introns 
has been constructed by cutting and pasting between pUNK54 and 
pUNK54delta1234.  Surprisingly all of these constructs so far tested 
are completely active.  This includes the plasmids pUNK54delta1, 
pUNK54delta234 pUNK54delta134, and a plasmid 'pUNK54deltam1+234', in 
which 389 b internal to the 562 b intron #1 have been deleted as well 
as introns 2,3,and 4.) In the cases of pUNK54delta1 and pUNK54delta234,
transformed lines have been obtained and exhibit wild type patterns 
of unc-54 tissue specificity.
The full activity of the four 'subset' plasmids has several 
consequences in terms of any explanation for why the introns are 
required.  First, the intron subset deletions were constructed in such 
a way that every segment of the inactive plasmid pUNK54delta1234 was 
subsequently recloned in generating at least one of the fully active 
construct.  Thus the inactivity of pUNK54delta1234 cannot be the 
result of some spurious mutation either present in the original 
synthetic cDNA construct or introduced during cloning.  Indeed these 
data indicate that no single sequence within the first four introns is 
absolutely required for efficient unc-54 expression.  Rather the 
requirement for 'something' within introns 1-4 can be satisfied at 
minimum either by the presence of the very short intron #2 (at 38 b.  
this is the shortest intron in unc-54), or by the trimmed intron #1 
present in pUNK54deltam1+234.  Although it is possible that this 
'something' is some sort of redundant enhancer or promoter element 
that is present in both of these segments, it seems quite possible 
that there is a mechanistic requirement for a splice near the 5' end 
of the long unc-54 message in order for the message to be expressed.  
Particularly puzzling if this were true would be the fact that even 
the pUNK54delta1234 plasmid still has four introns, and that the 
contiguous unspliced 5' region in pUNK54delta1234 is actually shorter 
than the longest contiguous region in the wild type unc-54 message (
exon #6).  Thus one might propose that certain worm messages need a 
splice somewhere near their 5' end.
Downstream Requirements: We have also undertaken a deletion analysis 
of the 3' end of the gene.  The sequence landmarks in the 203 nt 3' 
untranslated region of the message are an AAUAAA sequence (present 
upstream of most eukaryotic cleavage and polyadenylation sites), and 
the presumed the 3' end of the RNA (35 b.  downstream of the AAUAAA; 
suggested by sequencing of cDNA by A.  MacLeoud, S.  Brenner, and J.  
Karn).  Surprisingly neither of these sequences is necessary for 
function of the gene.  This is shown by the full activity, both in 
transient assays and in appropriate expression in transformed lines, 
of a clone deleting all of the 3' flanking region back to 63 b 
upstream of the AATAAA.  This construct has no AATAAA for at least 3kb 
downstream from the gene; a second construct replacing plasmid 
flanking sequences in this deletion with the sequences normally 3' to 
the unc-54 gene is also active.
There do seem to be some 'required' sequences within the 3' 
untranslated leader.  This is shown by a set of deletions which remove 
the entire 3' non-transcribed region back to 79, 108, and 116 nt 
upstream of the AATAAA.  These constructs are inactive, i.e.  they do 
not rescue unc54(e190) animals.  Thus some sequence between 63 and 79 
nt upstream of the AATAAA appears to be involved in proper expression 
of the gene.  This region lies in the center of various folded 
structures that can be drawn for the unc-54 3' leader.
Rock Pulak and Phil Anderson reported in the last gazette that a 
genetically isolated deletion (r293) in the 3' region of the 
endogenous unc-54 gene behaves as an unc-54 null.  It is tempting to 
speculate that the inability of the 79, 108, and 116 deletions to 
function is related to the inactivity of r293.  Pulak and Anderson 
also isolated and characterized suppressors of the r293 allele (which 
they and J.  Hodgkin have shown correspond to a set of morpho-mabs 
described in detail in the last Gazette by Hodgkin, Papp, and Ambros). 
At the suggestion of Anderson and Pulak, we tested whether the 
requirement for sequences between -63 and -79 from the AAUAAA could be 
suppressed by one of these mab mutants.  This was done by injecting 
the -79 deletion into unc-54(e1092)mab-1(e1228) animals.  The -79 
deletion is indeed active in these animals; a transgenic line PD107 
was derived, and the resulting transgenic locus loses the unc-54 
suppressing activity when Mab-1+ function is present.  The pattern of 
expression of unc-54 in PD107 (as well as in a number of the mab-
rescued r293 lines) is normal, suggesting that the identified 
sequences required in the the 3' leader probably do not play a role in 
tissue specificity.
Puzzle: Some sequence(s) within the unc-54 clone must be responsible 
for tissue specificity.  Either these are present within the coding 
sequences of the first three exons, in a short 3' region (~100nt) yet 
unexplored or redundant information is present so that no single 
sequence is necessary for tissue specificity.
[See Figure 1]

Figure 1