Comparison of the
mec-3 DNA sequences from C. elegans and WS9-6 ( another Caenorhabditis species) shows that all the exon sequences are conserved, and in addition, four blocks of nearly identical sequence are found upstream from the start codon (Way, WBG 11, 4). On the assumption that these conserved upstream regions are regulatory sequences, we have used in vitro mutagenesis to investigate their function. Specifically, we have altered various sites in these conserved regions in the
mec-3-lacZ fusion plasmid pTU28 (Way and Chalfie, Genes and Dev.3:1823) to see if the conserved sequences are important in determining when and where
mec-3 is expressed. Mutations in either Site II or Site III cause a defect in
mec-3 maintenance. A disruption of site II causes a severe maintenance defect: the
mec-3-lacZ fusion is expressed briefly after the expressing cells are born, then expression is lost for the rest of development. This pattern of expression is the same as when a non- mutant
mec-3-lacZ fusion is placed in a strain with a chromosomal mec- 3 mutation. A mutation in site III has a less dramatic effect: expression comes on in all the correct cells, then gradually fades, so that by adulthood, the number of expressing cells is significantly less than for a wild-type
mec-3-lacZ fusion. Within the conserved regions are putative binding sites for both mec- 3 and
unc-86 (see below and previous newsletters; the
mec-3 binding site in Site II is inferred largely from the mutant defect and is only weakly related to the ISL-1 footprint site). Because site II appears to have an
unc-86 binding site, we decided to test whether
mec-3 expression would depend on
unc-86 for maintenance as well as establishment. For ease of strain construction, we first made an integrated derivative of the
mec-3-lacZ/unc-22 (antisense) extrachromosomal element uEx4 by gamma-ray treatment (Kari et al. WBG 11, 3, p. 14); this integrated element is termed jeIn2. jeIn2 was placed in a temperature sensitive
unc-86 background (
n848), so that
unc-86 could be inactivated after
mec-3 expression had been established.
mec-3 does appear to require
unc-86 for maintenance. If jeIn2;
n848 is shifted to the non-permissive temperature after
mec-3-expressing cells are born, they lose expression of
mec-3. For example, if this strain is grown at 15 C until the mid-L1 stage, then either stained immediately or shifted to 25 C, the stained L1s express
mec-3-lacZ at normal levels. However, if the shifted animals are stained after the L4 stage, expression of
mec-3-lacZ is greatly reduced. [See Figure 1]