An account is given of the discovery in 1985 of the classical Cys2His2 (C2H2) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of subsequent structural studies on its 3D structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner structural hydrophobic core. This work showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of different lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that this zinc finger is found widespread in nature, in 3% of the genes of the human genome. It had long been the goal of molecular biologists to design DNA binding proteins for control of gene expression and we have adopted the zinc finger design and principle for this purpose. We demonstrated that the zinc finger design is ideally suited for such purposes, discriminating between closely related DNA sequences both in vitro and in vivo, and we have therefore adapted this natural design for engineering zinc finger proteins for targeting specific genes. The first example of the potential of the method was published in 1994 when a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In the same paper we also showed that we could activate a reporter gene by targeting a nine base pair promoter which we had inserted. Thus by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. By combining the targeting zinc fingers with other effector or functional domains e.g. from nucleases or integrases, to form chimeric proteins, genomes can be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of potential therapeutic importance.
A new-type of oxidation–reduction condensation using alkyl diphenylphosphinites 1 and 2,6-di-tert-butyl-1,4-benzoquinone (DBBQ) proceeded smoothly under mild and neutral conditions by using nucleophiles such as nitrogen-, carbon- or sulfur-ones whose pKa values were lower than 14. Various chiral molecules were successfully synthesized from chiral secondary or tertiary alkyl diphenylphosphinites in good to high yields with inversion of stereochemistry via SN2 displacement. Effects of the substituents of 1,4-benzoquinones and the pKa values of nucleophiles on these reactions have been investigated in detail.
Light scattered by assemblies of a feast/famine regulatory protein (FFRP), pot1216151 (DM1) from the hyperthermophilic archaeon Pyrococcus sp. OT3, was measured by combining an multi-angle detector with a gel filtration device. Four peaks were separated by gel filtration. For peak 4, the molecular weight (MW) was estimated as ~20K, a value not so different from that of dimers, 17K, by measuring static light scattering. For peak 3, MW was estimated as ~33K, suggesting formation of tetramers of MW of 34K. Peak 2 was the smallest among the four peaks, and not clearly separated from nearby peak 3, upon measuring light scattering. While, MW estimated for peak 1 was 112-118K, an intermediate between that of octamer, 69K, and that of hexadecamers, 138K. On the basis of these findings transition between assembly forms is discussed.