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We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations(More)
A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in(More)
Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine(More)
We used video assays to study the dynamic instability behavior of individual microtubules assembled in vitro with purified tau, purified MAP2 or a preparation of unfractionated heat-stable MAPs. Axoneme-nucleated microtubules were assembled from pure tubulin at concentrations between 4 and 9 microM in the presence of MAPs, and observed by video-differential(More)
The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules.(More)
Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The(More)
  • R A Walker
  • 1995
Motor domains of the Drosophila minus-end-directed microtubule (MT) motor protein ncd, were found to saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to ncd, mixtures of ncd motor domain and MTs were treated with the zero-length cross-linker(More)
We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the(More)
The Drosophila non-claret disjunctional (Ncd) kinesin-like protein is required for spindle assembly in oocytes and spindle maintenance in early embryos. Through the action of ATP-dependent microtubule (MT)-binding sites in the head and ATP-independent MT-binding sites in the tail, Ncd may bundle and, perhaps, slide MTs relative to each other. Our previous(More)
Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens(More)