Zeitler, Bryan, Gregory, Philip, Miller, Jeffrey, Lo, Te-Wen, Wood, Andrew, Zhang, Lei, Meyer, Barbara, Rebar, Edward, Schartner, Caitlin, Pickle, Catherine, Lee, Andrew, Urnov, Fyodor
[
International Worm Meeting,
2011]
Genome sequencing has facilitated research beyond traditional model organisms, but the paucity of broadly effective reverse genetic tools has limited cross-species comparisons of gene function needed to explore biological mechanisms. To overcome this limitation for nematodes, we developed a strategy for heritable, targeted gene disruption using engineered nucleases: fusions between custom-designed DNA binding domains of either the C2H2 zinc-finger motifs (ZFNs) or transcription activator-like effector (TALE) repeat motifs and the endonuclease FokI. ZFNs and TALE nucleases (TALENs) induce a double-strand break at a desired locus that can be imperfectly repaired to yield small insertions and deletions. Procedures were optimized for C. elegans (Ce) using ZFNs to recover mutant lines without reliance phenotype. TALENs proved equally effective, yielding the first TALEN-induced animal gene knockouts.
We applied this technology to C. briggsae (Cbr) to study the evolution of sex determination (SD) and X-chromosome dosage compensation (DC). The DC machinery and key components of the genetic hierarchy that regulate SD and DC proved to be functionally conserved over 15-30 Myr. In contrast, recruitment elements that target the DC machinery to X chromosomes have diverged. The Ce X motifs that are enriched on X relative to autosomes and pivotal for recruiting the Ce DCC to X are not enriched on the Cbr X. Moreover, all DCC recruitment elements imported from Ce into Cbr, fail to bind the Cbr DCC. ChIP-seq confirmed that DNA target specificity has diverged, and on going experiments will identify sequence specificity for Cbr DCC binding.
Like many developmental regulatory proteins (e.g. Twist, Dorsal), the DCC controls hundreds of genes through its action on cis-acting target sites. However, the evolution of DCC recruitment sites followed a very different pattern from that of binding sites for regulatory proteins that control multiple, unrelated developmental and cellular processes. Pleitropy of Twist and Dorsal, caused the proteins to accumulate few functionally significant changes to their DNA binding domains or their cognate DNA binding motifs. In contrast, DCC complex with multiple targets but lacking the constraints of pleiotropy exhibited a divergence of binding sites. Such divergence could have been an important driver for nematode speciation.
[
J Biol Chem,
2001]
Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.