Worm Breeder's Gazette 11(5): 53
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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 staining. 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.