Worm Breeder's Gazette 9(1): 56
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.
We have previously described a gut-localized esterase as a biochemical marker with which to study the early events in lineage- specific gene expression (Abstracts #14 and 86 in May, 1985, CSH; also Edgar and McGhee, Developmental Biology, in press). We wanted to find a second gut-specific hydrolase with which to compare the esterase expression and an acid phosphatase appears to be a good candidate. Adult worms are turned into extrudates by cutting in the presence of 2 g/ml of levamisole (see Lewis, et. al., Genetics 95: 905 (1980)), fixed in paraformaldehyde and stained with the following mixture: 50 l of 20 mg/ml of 1-naphthyl phosphate, 100 l of 1 M NaOH, 850 l 0.15 M sodium acetate to which is added 100 l freshly diazotized pararosaniline. Final staining pH is close to 5.0. Staining is usually rapid, intense and gut-localized. However, this staining pattern shows two important differences from the esterase pattern. Whereas esterase staining seems to occur throughout the cytoplasm of all gut cells, phosphatase staining appears localized to the edge of the gut lumen in the region of the brush border. (This same pattern is found in worms grown axenically and therefore does not result from clinging E. coli). Moreover, phosphatase staining is not found in the anterior six cells of the gut (i.e., those cells which derive from the anterior daughters of Ea(1/r)a and Ea(1/r)p cells and which show no later nuclear divisions). Since several cells at the posterior end of the gut also do not stain, there seems to be some symmetry in the phosphatase distribution but we re not yet certain how this correlates with their variable nuclear divisions. In any case, the staining pattern indicates that a spatial decision to express or activate the phosphatase is made relatively late in intestinal development. Under the usual staining conditions, the gut phosphatase appears to be the major phosphatase found in crude extracts of unsynchronized populations. Stained isoelectric focusing gels show one major band ( pI about 4.9) along with several minor bands. Unlike the esterase, the phosphatase is stable at its pI and gels can be left to stain overnight, allowing phosphatase activity to be detected in the equivalent of one worm or less. Preliminary steps in purification are consistent with the phosphatase being a membrane protein (i.e., activity is enhanced by Triton X-100) and Triton increases the apparent Molecular Weight on Sephacryl columns. Phosphatase activity in crude extracts is inhibited by tartrate but not by other phosphatase inhibitors such as alloxan, CuSO4, formaldehyde or EDTA. We will probably follow the same scheme worked out for the gut esterase, namely to purify the enzyme as a first step towards gene cloning and to induce isoelectric focusing mutants to locate the phosphatase gene on the genetic map.