Molecular Biology of the Cell · 2005 · 108 citations · 38 references
Life SciencesCell BiologyMolecular BiologyCytoskeletonGene ExpressionMorphogenesisActin FilamentsActin PolymerizationCytoskeletal Associated ProteinsEpithelial MorphogenesisIntestinal Barrier FunctionIntestinal DevelopmentActin MutationsC. Elegans Developmental ModelsFluorescent Reporter SystemsGreen Fluorescent Protein ReportersGut Microvilli StructureImmunofluorescence ImagingIntestinal Barrier FormationSecretory-excretory System
Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.
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Julie Thompson, Desmond G. Higgins, Toby J. Gibson · Nucleic Acids Research · 1994 · 64.9K citations · Full text
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
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