1 Departments of Genetics and Biochemistry and Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire
2 Department of Chemistry, Dartmouth College, Hanover, New Hampshire
3 Biomol Inc., Plymouth Meeting, Pennsylvania
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ABSTRACT |
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AMP-lysine hydrolase; W chromosome; dominant negative; site-directed mutagenesis
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INTRODUCTION |
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Although it is known that male birds are homogametic with a ZZ karyotype and females are heterogametic with a ZW karyotype, the molecular basis for sexual differentiation is unknown, although the existence of a ZZW female warbler strongly suggests that genetic information on the W chromosome is responsible for feminization (1). In birds other than ostriches and emus, the female-specific W chromosome carries 40 tandem repeats of an unusual Hint-related gene, ASW, whereas the Z chromosome carries a typical HINT gene (7, 12). In a striking departure from all previously isolated Hint homologous sequences, which conserve the AMP-lysine binding site more than other residues, the female-specific Asw protein has strong similarity to Hint except that 15 of 16 substrate-interacting residues are sexually dimorphic, i.e., altered in the W-encoded Asw with respect to the Z-encoded Hint (13). Thus, because the predicted dimerization interface (helix
2 and beta strand ß4) is virtually unaltered in Asw (13), and a single His-to-Ala substitution can reduce the catalytic activity of Hint by over 100,000-fold (2), evolutionary pressures may have ablated the AMP-lysine binding site in Asw but allowed Asw to function as a Hint heterodimerization partner. In this regard, it is important to note that of the 16 substrate-interacting residues in the Hint dimer, only one interacts with the AMP-lysine substrate across the dimer interface (8). That residue, Trp123, in the COOH-terminal Trp-Pro-Pro-Gly motif of Hint, is conspicuously substituted by Gln in the repeated, female-specific Asw sequence (13).
As a candidate female sex-determining gene, we reasoned that dominance and negativity might be functionally separable components of Asws activities. Negativity would seem to be a function of loss of AMP-binding residues in the Asw sequence, but, because the Hint dimer is not cooperative with respect to substrate hydrolysis (2), an inert dimerization partner would fail to depress Hint enzymatic activity by more than 50%. In fact, because there is no HINT gene on the W chromosome, HINT gene dosage is already reduced 50% by HINT gene absence. We therefore considered an explanation necessary for why a potential Hint dimerization partner lacking an active site is repeated 40 times on the W chromosome. By constructing a model of the putative Hint-Asw heterodimer based on crystal structures of rabbit Hint bound to products (3) and substrate analogs (8), we noted that Hint Trp123 is expected to be located on the Asw side of the dimer interface (13). More importantly, the model predicted that the residue in Asw that corresponds to Hint Trp123, namely Gln127, is physically located in the Hint half of the putative heterodimer. Thus we proposed that Gln127 in place of Trp123 is responsible for dominant depression and/or dominant alteration of activity of the Hint active site (13). In this study, with site-directed mutagenesis of chicken Hint and synthesis of a novel fluorescent Hint substrate, we establish that the W123Q allele of Hint creates a 2,700-fold alteration of substrate specificity, supporting a mechanistic role for Asws COOH-terminal Gln in feminization of developing birds.
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EXPERIMENTS AND RESULTS |
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DISCUSSION |
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This study is also instructive in dissection of how nature constructs a dominant-negative allele. As described by Ira Herskowitz (6), a dominant-negative allele ought to have two characteristics: simple loss of function and dominant interference. In the case of Asw, loss of function can be attributed to mutation of the active-site residues (13), which is sufficient to produce an inactive allele in vitro and in vivo (2). Because overexpression of an inert molecule would be pointless, however, we searched for the source of dominant interference in Asw. Transplantation of the dimer-crossing Trp to Gln substitution of Asw into the Hint sequence both depressed and altered specificity, consistent with a dominant-negative mechanism for Asw function in avian feminization.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Brenner, Dartmouth Medical School, Rubin 733-HB7937, Lebanon, NH 03756 (E-mail: charles.brenner{at}dartmouth.edu).
10.1152/physiolgenomics.00204.2004.
1 The Supplemental Material for this article (Supplemental Methods and Figs. S1S3) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00204.2004/DC1.
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REFERENCES |
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