Worm Breeder's Gazette 13(5): 58 (February 1, 1995)

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.

The heterochronic gene lin-28 is cloned

Eric G. Moss and Victor Ambros

Dept. of Biological Sciences, Dartmouth College, Hanover, NH 03755

      The heterochronic gene lin-28 is required for the normal timing and
synchrony of developmental events after the L1.  Mutations in lin-28 cause
the cell fates specific to the L2 in many lineages to be skipped. 
Consequently, L3-specific fates are expressed instead, L4- and adult-
specific fates are expressed precociously, and the animal ceases molting
after three larval stages.  lin-28 appears to interact with the
heterochronic gene lin-14, whose role in regulating developmental timing
is well established, but whose mechanism of action is completely unknown.
      lin-28 lies between mei-1 and ceh-6, and interval of about 0.25 map
units on LGI.  The yeast artificial chromosome Y37F9 was purified from a
CHEF gel using LMT agarose, agarase digestion, and a spin
microconcentrator, and injected into animals of the genotype lin-28
unc-29; nDp4.  Twenty stable transgenic lines were obtained and Unc
animals were examined for rescue.  One line displayed complete rescue of
lin-28 animals to Egl+.  Cosmids under this YAC were tested for rescuing
activity, and one cosmid, ZC550, displayed full rescuing activity.
      ZC550 was restriction-mapped and subcloned.  All of the subclones
that are able to rescue lin-28 overlap each other, and the smallest of
these is a 6kb PstI fragment.  The sequence of the rescuing fragment was
determined and was analyzed using Genefinder (thanks to Mark Metzstein)
and BLAST, and is likely to encode a gene that is a member of the Y-box
family of nucleic acid binding proteins, which share a 'cold shock domain'
with cold shock proteins of prokaryotes.  The filling in of a restriction
site in the cold shock domain-encoding sequence to create a 4 base
insertion, thus causing a frameshift in the open reading frame, rendered
the fragment unable to rescue a lin-28 mutant.  We sequenced the cold
shock domain from two lin-28 alleles, and one of them showed two missense
mutations.  In northern analysis, the rescuing fragment hybridizes to two
RNAs of 1.6kb and 1.5kb in length.  RT-PCR analysis indicates that the Y-
box gene RNA is expressed in L1 and not in L2.  L1 expression of lin-28 is
consistent with the genetic data indicating an interaction with lin-14. 
      The family of Y-box proteins, to which lin-28 seems to belong,
includes proteins that regulate gene expression by binding DNA or RNA,
including activators and repressors of transcription and repressors of
translation.  Our findings so far suggest that lin-28  may directly
regulate the expression of genes that implement L2-specific cell fates,
and may be expressed prior to the time that these fates are executed.