Zinc-finger proteins of the Cys 2 -His 2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structurebased modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up-or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.From the simplest of organisms to the most complex, transcriptional regulation is achieved primarily by proteins that bind nucleic acids. The advent of genomic sequencing and the availability of the complete sequences of several genomes provide new opportunities to study biology and to develop therapeutic strategies through specific modulation of the transcription of target genes. Of the DNA-binding motifs that have been manipulated by design or selection, the TFIIIA-related Cys 2 -His 2 zinc-finger proteins have demonstrated the greatest potential for manipulation into general and specific transcription factors (1, 2). Each Cys 2 -His 2 zinc-finger domain consists of approximately 30 amino acids and typically binds 3 base pairs of double-stranded DNA sequence (3, 4).Specific delivery of a DNA-binding protein to a single site within a genome as complex as that found in humans, 3.5 billion bp, requires an address of at least 16 bp. Statistically assuming random base distribution, a unique 16-bp sequence will occur only once in 4 16 or 4.3 billion nucleotides, roughly the same size of a human genome (3.5 ϫ 10 9 bp). An 18-bp address would be specific within 68 billion base pairs of sequence. The 18-bp address could be specified by a protein containing six zinc fingers if the periodicity of the protein domains could match that of the DNA over this extended sequence. An address of this length would be sufficient to uniquely specify any locus within all known genomes. Although natural proteins containing long polydactyl arrays of zinc-finger domains have been inferred from sequence, no zinc-finger proteins have been demonstrated to bind such a long, contiguous DNA sequence. Structural studies of the five-finger human glioblastoma (GLI) protein-DNA complex (5) and biochemical studies of the nine-finger protein TFIIIA (6, 7) have demonstrated that DNA binding in these polydactyl proteins is dominated by the interactions of a select few fingers. Sequence-specific binding of more than three contiguous zinc-fin...

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

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