Estimating the Age of the Common Ancestor of Men from the ZFY Intron

ware [J. Devereux, P. Haeberli, 0. Smithies, Nucleic Acids Res. 12, 387 (1984)]. The DNA sequences of theAARE1 andARE2 genes are deposited at GenBank (P25628 and U51 790, respectively). 18. KO-5' and KO-3' primers (GAGGGGACGAAAATTAGCCGCTATTAATTCTGGTATTGCCACCTAGACAAGAAGTAAACAGACACAGATGcaagagttcgaatctcttagc and CTATAAAGATTTAATAGCTCCACAGAACAGTTGCAGGATGCCTTAGGGTCGActacgtcgtaaggccgtttctgac, respectively; the lowercase lettering corresponds to the LEU2 gene) were used in a polymerase chain reaction (PCR) with the LEU2 gene as a template to produce the selectable yeast gene flanked by ARE2 gene sequences [A. Baudin, 0. Ozier-Kalogeropoulos, A. Denouel, C. Cullin, Nucleic Acids Res. 21, 3329 (1993)]. This was used to transform a derivative of yeast strain 5051, heterozygousforthearelANA allele. To identify integrants at the ARE2 locus, we performed PCR on genomic DNA from these strains using are2-5' (CATTGCAGTTACACGTGAATGC), are2-3' (TAGCTCCACAGAACAGTTGCAGG), and a 3' primer corresponding to the LEU2 gene (L2-3': CTCTGACAACAACGAAGTCAG). 19. P. Greenspan, E. P. Mayer, S. D. Fowler, J. Cell Biol. 100, 965 (1985). 20. One to two units (at an absorbance at 600 nm) of cells were incubated in YPD or defined media containing 1 ,uCi/ml of [3H]oleate in tyloxapol-ethanol (1:1) for 16 hours. Total lipids were prepared by hexane extraction [L. W. Parks, C. D. Bottema, R. J. Rodriguez, T. A. Lewis, Methods Enzymol. 111, 333 (1985)] and analyzed by thin-layer chromatography on DC-plastikfolien kieselgel 60 plates (EMerck, Germany). The plate was developed in hexane, diethyl ether, and acetic acid (70:30:1) and stained with iodine vapor. Incorporation of label into triglyceride and ergosterol ester was ascertained after scintillation counting and normalization to a [14C]cholesterol internal standard and the dry weight of the cells. 21. S. L. Sturley, H. Yang, J. T. Billheimer, in preparation. 22. To overexpress the ARE1 gene by copy number under the control of its own promoter in YEp3-16, a 2354-bp Cla fragment from pH3(34), encompassing the entire ARE1 gene, was blunt-ended with Klenow DNA polymerase and introduced into the Sma site of YEp352. To constitutively overexpress ARE1 from the ADH promoter in pADH536, a 2290-bp Nar fragment of pH3(34), starting 70 bp 5' to the ORF, was blunt-ended with Klenow and ligated to Klenow-treated, Eco RI-digested pDC-ADH [a derivative of pS5; S. L. Sturley et al. J. Biol. Chem. 269, 21670 (1994)]. Increased expression of the ARE1 transcripts, relative to that in a wild-type cell, was confirmed by Northern blot analysis. 23. C. C. Chang et al., J. Biol. Chem. 270, 29532 (1995). 24. G. J. Warner et al., ibid., p. 5772. 25. The incorporation of [1-'4C]acetate into saponified lipids was assessed as a measurement of sterol synthesis. Approximately 2 units at an absorbance of 600 nm of cells were incubated with 20 pLCi of [1 -14C]acetate in 2 ml of defined media at 300C for 3 hours and subjected to lipid saponification, hexane extraction, and thin-layer chromatography [R. Y. Hampton and J. Rine, J. Cell Biol. 125, 299 (1994)]. The incorporation of counts into total sterols was assessed after scintillation counting. To normalize the estimate of sterol biosynthesis to incorporation of acetate into the fatty acid pool, we acidified the aqueous lysate remaining after hexane extraction with concentrated HCI and re-extracted it with hexane [D. Dimster-Denk, M. K. Thorsness, J. Rine, Mol. Biol. Cell 5, 655 (1994)]. 26. I. Tabas, D. A. Weiland, A. R. Tall, J. Biol. Chem. 261, 3147 (1986). 27. M. Krieger and J. Herz, Annu. Rev. Biochem. 63, 601 (1994). 28. M. E. Basson, M. Thorsness, J. Rine, Proc. Natl. Acad. Sci. U.S.A. 83, 5563 (1986); S. L. Thompson, R. Burrows, R. J. Laub, S. K. Krisans, J. Biol. Chem. 262,17420(1987). 29. We gratefully acknowledge the assistance of 1. Becker, W. H. Mewes, and A. Goffeau in screening the confidential data set obtained in the European sequencing project. We thank L. A. Grivell and P. Philippsen for the provision of chromosome IlIl DNA clones and the shotgun library of cosmid 14-21 from chromosome XIV, respectively. We thank A. Keesler and 1. Tabas for helpful discussions, R. Golick for his assistance with confocal and fluorescence microscopy, N. Erdeniz for help with micromanipulations, and J. J. Rich, S. Gangloff, and A. Tinkelenberg for a critical reading of the manuscript. This work was supported in part by a Grantin-Aid/Investigatorship from the American Heart Association (New York City affiliate) and by the Ara Parseghian Medical Research Foundation to S.L. S. , NIH grants GM50237 and HG00861 to R.R. , R01 A138598 to M.B. , and HL40404 to R.J.D. , and by the European Community within the framework of the BIOTECH program. M.B. acknowledges support from the Johnson and Johnson Focused Giving program.