The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-functlon lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.
Bibliographical noteFunding Information:
Thanks to Alan Coulson and John Sulston for analyzing YAC, lambda and cosmid clones to identify overlaps with previously characterized clones, and also for their gift of gel purified YAC DNA and C.elegans genomic phage libraries. We are also grateful to Joan Park for providing the VT455 chromosome, to Diane Parry and H.R. Horvitz for providing lin-29(n1440) and to Robert Waterson for providing the gridded YAC library. Special thanks to Craig Mello, Diane Levitan and members of the laboratory for helpful discussions, and to Hugo Giambarella for technical assistance. This work was supported by Public Health Service research grant GM34O28 and American Cancer Society Grant NP479A (to VA), and by the Damon Runyon/Walter Winchell Cancer Fund (to AER). Some of the strains used in this work were provided by the Caenorhabditis Genetics Center, which is supported by Contact N01RR-4-2111 between the NIH Division of Research Resources and Curators of the University of Missouri.