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      The Caenorhabditis elegans paxillin orthologue, PXL-1, is required for pharyngeal muscle contraction and for viability

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          Abstract

          Caenorhabditis elegans pxl-1 is the orthologue of vertebrate paxillin and is expressed in body wall and pharyngeal muscle. In body wall muscle PXL-1 localizes to dense bodies, M-lines, and adhesion plaques, and in pharyngeal muscle PXL-1 localizes to podosome-like actin attachment complexes. PXL-1 is required in the pharynx for muscle contraction and viability.

          Abstract

          We have identified the gene C28H8.6 ( pxl-1) as the Caenorhabditis elegans orthologue of vertebrate paxillin. PXL-1 contains the four C-terminal LIM domains conserved in paxillin across all species and three of the five LD motifs found in the N-terminal half of most paxillins. In body wall muscle, PXL-1 antibodies and a full-length green fluorescent protein translational fusion localize to adhesion sites in the sarcomere, the functional repeat unit in muscle responsible for contraction. PXL-1 also localizes to ring-shaped structures near the sarcolemma in pharyngeal muscle corresponding to podosome-like sites of actin attachment. Our analysis of a loss-of-function allele of pxl-1, ok1483, shows that loss of paxillin leads to early larval arrested animals with paralyzed pharyngeal muscles and eventual lethality, presumably due to an inability to feed. We rescued the mutant phenotype by expressing paxillin solely in the pharynx and found that these animals survived and are essentially wild type in movement and body wall muscle structure. This indicates a differential requirement for paxillin in these two types of muscle. In pharyngeal muscle it is essential for contraction, whereas in body wall muscle it is dispensable for filament assembly, sarcomere stability, and ultimately movement.

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          Most cited references53

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          Multiple sequence alignment with the Clustal series of programs.

          R Chenna (2003)
          The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute) (http://www.ebi.ac.uk/clustalw/).
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            Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

            We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
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              Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

              The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                molbiolcell
                mbc
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                15 July 2011
                : 22
                : 14
                : 2551-2563
                Affiliations
                [1] aDepartment of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
                [2] bDepartment of Pathology, Emory University, Atlanta, GA 30322
                [3] cDepartment of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
                University of Bristol
                Author notes
                *Address correspondence to: Don Moerman ( moerman@ 123456zoology.ubc.ca ).
                Article
                E10-12-0941
                10.1091/mbc.E10-12-0941
                3135480
                21633109
                3f21b50e-e3b2-47b9-954e-b356c8cbc98a
                © 2011 Warner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

                “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

                History
                : 06 December 2010
                : 10 May 2011
                : 19 May 2011
                Categories
                Articles
                Cytoskeleton

                Molecular biology
                Molecular biology

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