Worm Breeder's Gazette 11(5): 22

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Cloning the Heterochronic Gene lin-4 by Polymorphism Mapping and Transformation Rescue

Rosalind C. Lee, Rhonda L. Feinbaum and Victor Ambros

Figure 1

We genetically mapped the lin-4 locus to a position closely linked 
and to the left of bristol/bergerac polymorphism maP13, detected by 
YAC Y42A4.  Using YAC and cosmid clones in the region as probes to 
southern blots, we analyzed DNA from the only lin-4 allele available 
at the time, e912, (see Feinbaum, et, al, this WBG issue) and found 
that the YAC Y18C1 detected a polymorphism in a 5 kb EcoRI band.  
Cosmids drawn under Y18C1 on the physical map did not detect this band,
so we suspected that lin-4 was at the right end of Y18C1, which 
extended into a cosmid-free gap.  Therefore, to clone the sequences 
corresponding to the 5 kb EcoRI band, we used Y18C1, which detects the 
5 kb RI band, and Y42A4, which almost completely overlaps Y18C1 but 
does not detect the band, in a differential screen of an EcoRI lambda 
library prepared by Ann Rougvie.  In this way we isolated two 
different 5 kb EcoRI fragments that both turn out to be partially 
deleted in lin-4(e912) DNA.  Much to our surprise, these clones lie 
not in the gap, but in the contig and to the left of the previous end 
of Y18C1.  We have therefore revised the extent of Y18C1 to include 
the cosmids containing these two 5 kb EcoRI fragments.
One of the polymorphic 5 kb EcoRI clones, pRL2D, rescues the mutant 
phenotype of lin-4(e912).  (Two overlapping cosmids, C02B6 and C31G11, 
also rescue lin-4).  Therefore, pRL2D contains a functional lin-4 gene,
although the lin-4(e912) lesion, originally generated by [32P] decay, 
is somewhat complex, involving deletion of another 5 kb EcoRI fragment 
in the region, and partial duplication of a 3.5 kb fragment.  We are 
currently sequencing pRL2D and testing smaller subclones for rescue.

Figure 1