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      Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae

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          Abstract

          To determine how microtubule (MT) nucleation and nuclear migration are controlled in multinucleated hyphae we deleted genes encoding MTOC subunits and AgStu2. The novel phenotypes we observed in these mutants compared with analogous deletions in budding yeast allowed us to assign functions to the two types of cMTs that we observe in A. gossypii.

          Abstract

          In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.

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          Molecular Cloning : A Laboratory Manual

          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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            The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome.

            We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.
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              Closing mitosis: the functions of the Cdc14 phosphatase and its regulation.

              Completion of the cell cycle requires the temporal and spatial coordination of chromosome segregation with mitotic spindle disassembly and cytokinesis. In budding yeast, the protein phosphatase Cdc14 is a key regulator of these late mitotic events. Here, we review the functions of Cdc14 and how this phosphatase is regulated to accomplish the coupling of mitotic processes. We also discuss the function and regulation of Cdc14 in other eukaryotes, emphasizing conserved features.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                mbc
                mbc
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                1 March 2010
                : 21
                : 5
                : 753-766
                Affiliations
                [1]*Department of Molecular Microbiology, Biozentrum University of Basel, 4056 Basel, Switzerland;
                [2] Stowers Institute for Medical Research, Kansas City, MO 64110; and
                [3] Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160
                Author notes
                Address correspondence to: Sue L. Jaspersen ( slj@ 123456stowers.org ).
                Article
                3566656
                10.1091/mbc.E09-07-0555
                2828962
                20053682
                cdfed360-3519-43f4-9104-f9da6317a58e
                © 2010 by The American Society for Cell Biology
                History
                : 8 July 2009
                : 21 December 2009
                : 24 December 2009
                Categories
                Articles
                Cytoskeleton

                Molecular biology
                Molecular biology

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