Worm Breeder's Gazette 12(5): 35 (February 1, 1993)

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.

A lac-Z Fusion Expressed in the C Lineage

Geraldine Seydoux, Andy Fire

Carnegie Institution, Baltimore MD 21210

During the course of our screen for embryonic expression patterns (WBG 12#4, p20 ),we have isolated a lac-Z fusion that appears to be expressed specifically in the C lineage. This fusion (pGS 15.02) consists of a 3.2 kb fragment of C. elegans genomic DNA fused to the lacZ gene in the pPD21 .28vector. We found that 1 kb of the 3.2 kb is sufficient to drive C specific lac-Z expression (pGS 15.24) and have used three lines carrying an integrated array of pGS15 .24to characterize its expression pattern.

To facilitate the identificadon of the lac-Z expressing cells, we have developed a modified method to stain lineaged embryos (thanks to Lois Edgar and Deborah Cowing for their helpful suggestions). Briefly, 2 to 4 cells embryos are transfered in a drop of modified lacZ staining mix (see below) onto a 1.5% agarose pad (also containing modified staining mix) and covered by a coverslip for Nomarski observadon. Embryos are allowed to develop and their lineage is recorded until a desired stage is reached (see WBG 12#2, p 19 for a descripdon of our recording system). At that time, the egg shell and vitelline membrane are disrupted using a laser microbeam to allow the staining mix to enter the embryo. 12 hours later, cells staining for lacZ can be idendfied based on their lineage idendty.

In this manner, we found that in the 28 cell-stage embryo the four C-derived blastomeres (Caa, Cap, Cpa, Cpp) all express the fusion in their cytoplasm. (In nonlineaged embryos, we sometimes saw two lacZ-expressing cells in 24 to 26 cellembryos, suggesting that Cp and Ca may occasionally start expressing the fusion towards the end of their cell cycle). The four C-derived blastomeres, as well as their 8 progeny continue to express the fusion during their endre cell-cycle. By the 16 C-cellstage (172 cells), the expression of the fusion weakens but still remains confined to the C lineage. During morphogenesis, no expression of the fusion has been observed in lines carrying the integrated array. Curiously, lines carrying pGS15 .24on an extrachromosomal array continue to express the fusion up to the comma stage. At this dme, expression of the fusion is seen in the nuclei of C-derived hypodermal cells. (We note, however, that expression of the fusion appears somewhat deregulated in non-integrated lines: in the 28 cell-stage embryo, many more cells besides the C-derived blastomeres often express the fusion.)

Sequencing pGS15 .24,we have found that it encodes a translational fusion between a novel C. elegans gene and lacZ. We have obtained and sequenced cDNAs corresponding to this gene and found that it has the potential to encode for a 38 kD protein containing an acidic region rich in glutamic acid. No homologies or other features have been noticed as of yet. By Northern analysis, this gene appears to be expressed predominantly in embryos, with no detectable expression in larvae and a low level of expression in adults.

Modified Staining Mix:

O.O5M NaPi pH 7.5

2mM M gCl2

5mM KFe/5mM KFo

1 ug/ml DAPI

0.004% SDS

5% Sucrose

0.125% X-gal

1% glutaraldehyde