Worm Breeder's Gazette 12(4): 19 (October 1, 1992)
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 begun to use the data generated by the C. elegans genome sequencing project to investigate the expression patterns of genes predicted therein. Sequence data in the C. elegans database, ACeDB, were used to design reporter-gene fusions between predicted genomic ORFs and lacZ Specific DNA fragments from sequenced cosmids were sub-cloned into one of Andy Fire's vectors, pPD21 .28, pPD22 .04or pPD22 .11(1).Each construct typically includes ~5kb from upstream of the predicted transcribed region, with ß-galactosidase expression in C. elegans being dependent upon translational fusion to lacZ After transformation of N2 with each construct, plus the dominant marker rol-6 ( su1006 )in pRF4 ,the expression pattern of the predicted gene was assayed by staining of transformants for ß-galactosidase activity. None of the transformants so far assayed have constructs integrated into the genome.
To date, 6 ORFs have been analyzed:
(i) ZK643 .1-Expressionwas seen in neural cells: 4 in the anterior ganglion, 8 in the lateral/ventral ganglia, with a further 46 in the tail ganglia. Staining was also seen on sub-cellular "particles" in the vulval muscle cells. The neural pattern develops from the L1 larval stage onwards; the vulval staining occurs from L4 onwards.
(ii) ZK643 .2-Generalstaining of embryos. (Previous examples of this kind of staining have been attributed the use of pRF4 as the transformation marker 2.)
(iii) ZK643 .3-Thenuclei of 4 of the vulval muscle cells stain up, as do body wall muscles in the head. Vulval staining can be seen in L4 -youngadult, with the head muscles staining earlier at ~L2-L4.
(iv) ZK643 .5-3 nuclei of the lateral/ventral ganglia show expression from L2 /L3onwards. Further staining of 3 cells, possibly in the tail ganglia, develops in adult worms along with a few nuclei in the ventral cord.
(v) F59B2 .5-Noexpression was detected.
(vi) F59B2 .13-Adultsshow expression in 4 nuclei of the lateral/ventral ganglia, in 2 large nuclei in the mid-body of the worm adjacent to either hypodermal ridge- one close to the vulva and one anterior to it- and in 2 nuclei (possibly neuronal) in the tail. The lateral/ventral ganglia staining was present at hatching, the mid-body and tail cells started to stain at later larval stages. Also seen in adults, but only after overnight incubation, are several (~4 typically) body-wall muscle cells in the posterior half of the animal.
4 out of 6 of the constructs assayed so far have produced specific staining. The preponderance of nerve cell staining may be a consequence of the few predicted ORFs so far examined. We hope to see a wider range of expression patterns revealed as further ORFs are analyzed in this way.
2. Andrew Fire and Susan White Harrison. WBG I 1:5 67.