Structural and functional characterization of the yeast general transcriptional activator CCR4

Transcription of the glucose-repressible ADH (ADH2 locus) in Saccharomyces cerevisiae is controlled by two regulatory pathways. The general transcriptional factors CCR4, CRE1, and CRE2 constitute the first pathway while the second pathway is comprised of the trans-activators ADR1 and CCR1. Both ADR1 and CCR1 act through upstream activation sequences (UAS) found in the 5\sp\prime-regulatory region of the ADH2 structual gene. In contrast, the action of CCR4, CRE1, and CRE2 is likely to be at sequences near the TATAA element of ADH2. The CCR4 locus was precisely mapped on the left arm of chromosome I where it had previously been localized. Plasmid constructions bearing sequences from chromosome I in the vicinity of CCR4 were tested for their ability to complement a defective ccr4 allele. A functional copy of CCR4 was identified and the DNA sequenced to reveal a 2511-bp open reading frame that predicts a protein with an estimated mass of 94.6-kDal. The CCR4 protein showed similarity with a family of proteins containing a leucine-rich tandem repeat motif. The repeats are characterized by the 24 amino acid repeating sequence P-X-X-o-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-o (where X = any residue; o = aliphatic residues L, I, or V) and have been suggested to represent a domain involved in protein binding. Deletion analysis of CCR4 indicates that these repeats are required for its proper function. The amino terminus of CCR4 showed similarities to a variety of transcription factors including TFIID, the TATAA binding factor from humans and Drosophila. Additional studies indicated that CCR4 mRNA and protein levels were not regulated by carbon source availability or the allelic state of the CRE genes. These results suggest that the interactions observed between CCR4 and the CRE genes occur directly or indirectly at a protein level. The possible role that CCR4 plays in the transcriptional regulation of the ADH2 locus based on (1) the sequence similarities seen between CCR4 and other proteins and (2) the functional characterization of deletions and disruptions created within the coding sequences of CCR4 are discussed

Frequency fluctuations in silicon nanoresonators

Frequency stability is key to performance of nanoresonators. This stability is thought to reach a limit with the resonator's ability to resolve thermally-induced vibrations. Although measurements and predictions of resonator stability usually disregard fluctuations in the mechanical frequency response, these fluctuations have recently attracted considerable theoretical interest. However, their existence is very difficult to demonstrate experimentally. Here, through a literature review, we show that all studies of frequency stability report values several orders of magnitude larger than the limit imposed by thermomechanical noise. We studied a monocrystalline silicon nanoresonator at room temperature, and found a similar discrepancy. We propose a new method to show this was due to the presence of frequency fluctuations, of unexpected level. The fluctuations were not due to the instrumentation system, or to any other of the known sources investigated. These results challenge our current understanding of frequency fluctuations and call for a change in practices

Continuous evolution of B. thuringiensis toxins overcomes insect resistance

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects

Binary Toxins from Bacillus thuringiensis Active against the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest