Worm Breeder's Gazette 11(5): 53

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unc-31 Update

David Livingstone

The smallest unc-31 fragment with which I have been able to obtain 
rescue is 13.5kb in length.  This fragment gave a smaller number of 
rescued animals than the cosmid, and many of these were only partially 
rescued.  It is possible that some sequences outside this fragment are 
necessary for the normal functioning of the gene.
The complete sequence of the rescuing fragment has been determined.  
The closing of the final gap in the sequence revealed the presence of 
a third U5-snRNA.gene (see WBG vol.  11 No.  2).  Overlapping cDNA 
fragments from the Ahringer and Maruyama cDNA libraries, together with 
two PCR amplified cDNA fragments, define a single transcript of at 
least 4kb.  The 3' end of the transcript has not been defined by any 
cDNA clone, but an attempt has been made to predict the splicing 
pattern of this part of the gene from genomic sequence.
The predicted unc-31 transcript encodes a protein of approximately 
120kd.  The sequence has no hydrophobic stretches of amino acids which 
look like signal or transmembrane sequences and, as far as I can tell, 
has no significant sequence similarity to other proteins.
I have used  -galactosidase translational fusion constructs and anti 
unc-31 antisera to study the expression pattern of the gene.  I have 
made three different  -gal constructs.using Andy Fire's vectors.  All 
three of these contain only the 5' end of the unc-31 gene.  The unc-54 
sequences present in the vector are used for transcript termination.  
Two of the constructs contain a fairly small amount of unc-31 upstream 
sequence, while the third has a much longer sequence upstream of the 
gene.  The two short constructs gave the same staining pattern.  There 
was strong staining of a small number of neurons in the head.  This 
pattern was somewhat variable, but most worms had the same set of five 
staining cells.  Some worms showed faint staining of a larger number 
of neurons.  There was also staining of posterior gut in L1s, and 
variable staining of non neuronal pharyngeal cells.  The variability 
of the staining, and the difficulty of explaining how the unc-31 
phenotype could result from defects in these cells, made it seem 
unlikely that this expression pattern was correct.  A third construct 
with a much longer upstream sequence was made.  This gave a 
qualitatively different staining pattern.  There were now a large 
number of strongly staining neurons, with other neurons staining 
faintly.  In strongly stained worms, all, or nearly all, neurons 
stained.  The relative intensity of staining of different neuron types 
seems to be at least partially reproducible between different 
individual worms, and different injected lines.  There is little or no 
staining of non-neuronal cells.  Some strains which resulted from co-
injection of the unc-31 construct with the twitcher plasmid, pPD10.46, 
showed variable staining of muscle cells, but there was little or no 
staining of non-neuronal cells when the construct was co-injected with 
other markers.  There still seems to be a small amount of variability 
in staining which cannot be explained by mosaicism.  It is possible 
that this is simply a result of the construct being present in the 
form of extrachromosomal arrays, but it could mean that it still lacks 
some sequences necessary for normal expression.  No integrated lines 
have been obtained with this construct.
In one experiment the larger  -galactosidase construct was co-
injected into unc-31;e928 worms with the unc-31 cosmid, C14G10.  Some 
of the rescued worms from this experiment had an odd behavioral 
phenotype.  The worms were egg laying constitutive.  They retained 
very few eggs in the uterus and, continued to lay eggs when removed 
from food.  They were also slightly Unc, but did not look like partial 
unc-31 rescues.  I am now looking to see if they have any other 
behavioral defects.  I do not know.if the egg laying constitutive 
phenotype is caused by the cosmid or the fusion construct, but am now 
looking at worms injected with each DNA alone to find out..  There are 
many possible explanations for the observed phenotype.  The most 
interesting are that it could result from overexpression, or ectopic 
expression, of the unc-31 gene.
Two fusion proteins, each containing about 33kdal of unc-31 sequence,
have been used to make polyclonal antibodies.  Antisera suitable for 
immunofluorescent staining of worms have been raised against both 
proteins.  The two antisera give the same staining pattern.  Wild type 
worms showed faint staining of the nerve ring, dorsal cord and ventral 
cord.  No staining of cell bodies could be seen, so it was impossible 
to tell which cells were staining.  The fact that the whole nerve ring 
appears to stain suggests that a large number of neurons are 
expressing the protein.  The failure to see cell bodies is probably 
significant.  The protein is located in cell processes.
The staining seen in N2 was reduced, or abolished, in the unc-31 
mutant ,e928.  This strain has a small deletion within the unc-31 gene.
Unc-31 worms carrying extrachromosomal arrays of the unc-31 cosmid, 
C14G10, stained much more strongly than wild type.  In some of these 
worms staining was strong, but is still mostly confined to cell 
processes.  Where the staining was even stronger, cell bodies stained 
brightly.  The staining pattern of these worms gives the best 
indication to-date of the expression pattern of the gene.  I have not 
had long to study the pattern, but it seems that all neurons stain to 
some extent.  Some neuron types.stain much more brightly than others.  
The.pattern is very similar, but not identical to the  -galactosidase 
The nerve ring staining was not seen in the pre-immune serum.  The 
preimmune serum of different animals did, however, give several 
different staining patterns.  One pattern, which may be of interest, 
was seen using the pre-immune serum of three different rats.  The 
serum from these rats stains a subset of neurons which look as if they 
are the cells reported to contain GABA.  The staining is bright, with 
cell bodies and processes clearly visible.
These results are still rather preliminary.  It would be very useful 
to improve the staining of wild type worms.  I have not had much time 
to optimize the staining procedure.  In would also be informative to 
locate the position of the protein within the cells more accurately.  
Immuno-E.M.  is a possibility.