Worm Breeder's Gazette 11(5): 49

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Sequences for Maintenance of mec-3 Expression

Jeff Way and Jie Zhang

Figure 1

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 
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]

Figure 1