Worm Breeder's Gazette 15(1): 80 (October 1, 1997)
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.
| 1 | Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 |
| 2 | Department of Biochemistry, University of Minnesota, St. Paul, MN 55108 |
The heterochronic genes lin-4, lin-14, lin-28 and lin-29 control the
relative timing and sequence of many events during post-embryonic
development, including the terminal differentiation of lateral
hypodermal seam cells. In the course of performing a variety of
genetic screens aimed at identifying additional members of the
heterochronic gene pathway(1) we have isolated four alleles of
lin-42, a heterochronic gene for which a single allele has been
reported(2). lin-42(n1089) mutants execute seam cell terminal
differentiation precociously, during the L3-to-L4 molt. Epistasis
analysis places lin-42 downstream of lin-4 and upstream of lin-29, a
position shared by lin-14 and lin-28. We set out to clone lin-42 to
better understand its role in the heterochronic gene pathway.
Because lin-42 resides in a gene-poor region of the left arm of
LGII, we elected to use a transposon tagging approach to facilitate
its cloning. We modified the gfp-based screen of Abrahante et al.(1)
to allow use of transposons as a mutagen. We constructed the strain
lin-4; mut-6; veIs13 [col-19::gfp + rol-6(su1006)]. These animals do
not express the col-19::gfpfusion (expressed adult-specifically in
wild type) due to the lin-4 mutation that blocks the execution of the
larval-to-adult switch in the hypodermis. We then screened for
restoration of col-19::gfp expression caused by Tc1-transposition
events. At least two of more than 35 mutations isolated from this Tc1
screen are additional lin-42 alleles. We detected a restriction
fragment length polymorphism in DNA isolated from one of these, ve27,
using Tc1 sequences as probe. The polymorphic fragment was cloned and
the DNA sequence flanking the Tc1 element was determined. Data base
searches with this sequence showed identity to cosmid F47F6, a
sequenced cosmid from LGII residing between RFLPs veP2 and nP48 in the
position where we had previously mapped lin-42. The Tc1 insertion
site disrupts the fourth exon of a Genefinder predicted gene, F47F6.2.
We have confirmed that this predicted gene corresponds to lin-42. An
8.9 kb clone containing only this predicted open reading frame and
approximately 3 kb 5'- and 1 kb 3'-flanking sequence rescues the
lin-42 mutant phenotype. In addition, we have sequenced several
lin-42 alleles and found lesions in Exons 2, 3, 4 and 5. We used
RT-PCR to determine the structure of the lin-42 mRNA, and found the
Genefinder prediction to be accurate.
Analysis of the predicted lin-42 protein using BLAST revealed
similarity to the PAS domain found in the family of proteins related
to Drosophila Period (PER). The PER PAS domain has been shown to
mediate protein-protein contact(3). LIN-42 shares highest identity
with the PASB domain in the Per-like protein from our friend, the
American cockroach Periplaneta americana. We are intrigued by the
presence of the PAS domain in the heterochronic protein LIN-42 and in
a variety proteins that time circadian rhythms including the PER
family, the white collar proteins of bread mold(4), and the CLOCK
protein of mice(5). This conservation of the PAS domain may reflect a
molecular link between proteins that control timing in worms and other
organisms. Expression studies are underway to test if the lin-42 mRNA
or protein products fluctuate during development. It is also possible
that the sharing of this domain between the heterochronic and
circadian rhythm classes of timing proteins simply reflects a
conserved protein interaction domain. We note that PAS domains
are also found other proteins, such as the aryl hydrocarbon receptor,
where they also serve a role in protein-protein contact. Many of
these proteins are transcription factors that include a basic
helix-loop-helix domain, apparently lacking from PER and LIN-42.
(1)Abrahante and Rougvie (1995). WBG 14, #1: 91; Abrahante, Miller and
Rougvie, Manuscript in preparation.
(2)Liu, Z. (1990). Ph.D. Thesis, Harvard University, Cambridge, MA.
(3)Nambu et al. (1991) Cell 67:1157; Huang et al. (1993). Nature
364:259.
(4)Crosthwaite et al. (1997). Science 276: 763.
(5)King et al. (1997). Cell 89: 641.