daf-16 : An HNF-3/forkhead Family Member That Can Function to Double the Life-Span of Caenorhabditis elegans
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C. Kenyon in C. elegans II D. R. Riddle T. Blumenthal B. Meyer J. Priess Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1997) pp. 791–814.
D. Riddle and P. S. Albert in C. elegans II D. R. Riddle T. Blumenthal B. Meyer J. Priess Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1997) pp. 739–768.
The process of dauer formation is facilitated by high temperature; therefore many daf-2 mutations induce dauer formation at high but not low temperature. At low temperature (or when shifted to high temperature as young adults past the dauer decision point) these daf-2 mutants become long-lived adults (4).
Because dauers are long-lived one explanation for these two roles is that weak daf-2 mutations allow adults to express only one feature of the dauer namely its slow rate of aging.
daf-2(e1370ts) L4-stage animals were mutagenized with trimethylpsoralen followed by UV irradiation (13) and F 2 progeny were screened for their ability to grow to adulthood at 25°C. Twenty-four independent daf-2 suppressers mu84 to mu107 were isolated all of which proved to be daf-16 alleles. We performed complementation tests by crossing daf-16(m26); daf-2(e1370); him-5(e1490) males with sup; daf-2(e1370) hermaphrodites at the nonpermissive temperature and examining cross progeny for dauer formation. The descendants of the cross progeny were also examined to ensure that the mutations were not unlinked noncomplementing mutations. We also mapped many of the mutations by testing for linkage with unc-29 which maps near daf-16 or else with daf-16 –linked restriction fragment length polymorphisms using PCR (32).
One such gene may be daf-18. This gene has been identified by a single mutation that suppresses both dauer formation and the life-span extension of daf-2 mutants (7 10 28). However many daf-18 individuals show severe morphological abnormalities suggesting that this gene has other possibly essential functions (28). The fact that we did not find any daf-18 alleles supports this hypothesis. In addition we note that because we screened F 2 progeny of mutagenized animals we would have missed mutants that were maternally rescued.
We first attempted to clone daf-16 by positional mapping but found that the gene was located in a gap in the physical map between cosmids AE7 and ZK39. To isolate daf-16::Tc1 insertion mutants we screened daf-2(sa189); mut-6 animals for spontaneous mutants that did not become dauers when cultured at 20°C. One mutant mu147 also suppressed dauer formation at 25°C. This mutation failed to complement daf-16(m26) and was closely linked to unc-29 which maps near daf-16. mu147 was subsequently crossed to either unc-29(e1072); daf-2(e1370) ; him-5(e1490) or daf-2(e1370) ; him-5(e1490) mutants and homozygous Daf-16(−) and Daf-16(+) recombinants were obtained. Genomic DNA was prepared from these recombinants and analyzed by Southern blot hybridization with the 1.6-kb Tc1 sequence as probe. A 6.1-kb Tc1-hybridizing fragment was detected in the Xba I–digested genomic DNA which was present in 20 of 20 daf-16 (−) recombinants but absent in 15 of 15 daf-16 (+) recombinants and also absent in the wild-type strain (N2). DNA from the corresponding region was then extracted from agarose gels and circularized by self-ligation. An inverse PCR strategy was used to identify a Tc1-containing fragment with the expected size of 5.1 kb. The Tc1-specific primers used for inverse PCR were 5′-CCTTGTTCGAAGCCAGCTACAATGGC-3′ and 5′-TGATCGACTCGATGCCACGTCGTTGT-3′. The 5.1-kb PCR product was cloned into the pGEM-T vector (Promega Madison WI) and the 0.6-kb flanking Tc1 sequence was removed by digestion with Eco RV. The remaining sequence was then used as a probe in subsequent experiments.
R. H. A. Plasterk and H. G. A. M. van Luenen in C. elegans II D. R. Riddle T. Blumenthal B. Meyer J. Priess Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1997) pp. 97–116.
Wilson R., et al., ibid. 368, 32 (1994).
RT-PCR was performed with an SL1 primer to obtain the 5′ end of the gene and with Q T Q O and Q I to obtain the 3′ end by rapid amplification of cDNA ends (RACE) as well as with several internal primers (positioned as shown in Fig. 2B). In addition blast searches with sequences contained on R13H8 identified three cDNA clones (yk13f11 yk31f10 and yk32f8) in a C. elegans EST database . Complete sequences of clones yk13f11 and yk31f10 were then obtained and both contained the longer form of the transcripts whereas the majority of the RT-PCR products (with mixed-stage RNA preparations) contained the shorter form (see Fig. 2). In addition the longer spliced form was also detected by RT-PCR.
K. Lin J. Dorman A. Rodan C. Kenyon data not shown.
P. Flakoll M. G. Carlson A. Cherrington in Diabetes Mellitus D. LeRoith S. I. Taylor J. M. Olefsky Eds. (Lippincott-Raven Philadelphia PA 1996) pp. 121–131.
R. M. O'Brien and D. K. Granner in ibid. pp. 234–241.
R. M. O'Brien et al. Mol. Cell. Biol. 15 1747 (1995).
Murakami S., Johnson T. E., ibid. 143, 1207 (1996).
Neither gain of function ( n1046) nor dominant negative ( sy100) mutations in the C. elegans Ras homolog let-60 affected C. elegans life-span (because these mutants cannot lay eggs their gonads were ablated to prevent premature death from internal hatching). In addition let-60(n1046gf) did not suppress the dauer-constitutive phenotype of daf-2(e1370) (J. Apfeld and C. Kenyon unpublished data).
C. E. Finch Longevity Senescence and the Genome (Univ. of Chicago Press Chicago IL 1990).
Frohman M. A., Methods Enzymol. 28, 341 (1993).
Ogg S., et al., ibid. 389, 994 (1997).
We thank N. Ahmada for technical assistance with positional mapping (16); B. Albinder M. Macrae J. Reiter T. Wang D. Eisenstadt I. Reichardt and J. Blumstein for helping to isolate and characterize trimethylpsoralen- and Tc1-induced daf-16 mutants; J. Apfeld in our laboratory for investigating the role of Ras in the daf-2 signaling pathways (30); members of the Kenyon lab for discussions and comments on the manuscript; and Y. Kohara for sending us EST clones of daf-16 . Supported by NIH grant AG11816. C.K. is the Herbert Boyer Professor of Biochemistry and Biophysics.