A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin–CDK complex

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This ‘CHH’ MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2–Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

In vivo evidence for a cochlear amplifier in the hair-cell bundle of lizards

Vertebrate sensory hair cells achieve high sensitivity and frequency selectivity by adding self-generated mechanical energy to low-level signals. This allows them to detect signals that are smaller than thermal molecular motion and to achieve significant resonance amplitudes and frequency selectivity despite the viscosity of the surrounding fluid. In nonmammals, a great deal of in vitro evidence indicates that the active process responsible for this amplification is intimately associated with the hair cells' transduction channels in the stereovillar bundle. Here, we provide in vivo evidence of hair-cell bundle involvement in active processes. Electrical stimulation of the inner ear of a lizard at frequencies typical for this hearing organ induced low-level otoacoustic emissions that could be modulated by low-frequency sound. The unique modulation pattern permitted the tracing of the active process involved to the stereovillar bundles of the sensory hair cells. This supports the notion that, in nonmammals, the cochlear amplifier in the hair cells is driven by a bundle motor system.

Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

Dendrimer-supported combinatorial chemistry.

A new methodology for the construction of combinatorial libraries is described. The approach, termed dendrimer-supported combinatorial chemistry (DCC), centers on the use of dendrimers as soluble supports. Salient features of DCC include solution phase chemistry, homogeneous purification, routine characterization of intermediates, and high support loadings. To demonstrate the feasibility of DCC, single compounds and a small combinatorial library were prepared via the Fischer indole synthesis. Excellent product yields and purities were obtained, and dendrimer-protected intermediates could be routinely analyzed by 1H and 13C NMR and by mass spectrometry. The results indicate that DCC is a general and efficient strategy for the generation of combinatorial libraries.