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Content Provider | World Health Organization (WHO)-Global Index Medicus |
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Author | Williams, B. D. Rosenbaum, J. L. Velleca, M. A. Curry, A. M. |
Description | Author Affiliation: Williams BD ( Department of Biology, Yale University, New Haven, Connecticut 05611.) |
Abstract | Chlamydomonas reinhardtii flagellar motility mutant pf-14 fails to assemble radial spokes because of a deficiency for assembly-competent radial spoke protein 3 (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck. 1981. J. Cell Biol. 88:80-88). Here, we raise an antiserum to protein 3 and use it to isolate the corresponding structural gene from an expression library. Southern blot analysis indicates that the gene is single copy and has not undergone major rearrangement in mutant pf- 14 cells. Northern blot analysis suggests that wild-type amounts of an apparently normal 2.3-kb transcript accumulate in mutant cells during flagellar regeneration. When this mutant RNA is hybrid selected and translated in vitro, however, it produces a slightly truncated polypeptide 3 with an altered charge. The mutant protein 3 fails to assemble into pf-14 flagella and is maintained within a cytoplasmic pool of unassembled radial spoke polypeptides, as indicated by immunoblot analysis of proteins from whole cells and isolated axonemes using antisera to several radial spoke polypeptides. Interestingly, amounts of the mutant protein are greatly diminished relative to other spoke components. Complete genomic and cDNA nucleotide sequences were determined, and the pf-14 mutation was identified. It is a C-to-T transition near the 5' end of the protein coding region, which changes codon 21 to the ochre termination signal UAA. The size and charge of the mutant protein, and its reduced levels in cells, suggest that it is produced by relatively inefficient translational initiation as codon 42. The unphosphorylated isoform of radial spoke protein 3 is identified, and the sequence similarities between intervening sequences of the radial spoke protein 3 gene and a conserved intervening sequence of the two Chlamydomonas beta-tubulin genes (Youngblom, J., J. A. Schloss, and C. D. Silflow. 1984. Mol. Cell. Biol. 4:2686-2696) are reported. |
ISSN | 00219525 |
e-ISSN | 15408140 |
Journal | The Journal of Cell Biology |
Issue Number | 1 |
Volume Number | 109 |
Language | English |
Publisher | Rockefeller University Press (United States) |
Publisher Date | 1989-07-01 |
Publisher Place | United States |
Access Restriction | Open |
Subject Keyword | Bacterial Proteins Chlamydomonas Genetics Flagella Physiology Proteins Protozoan Proteins Alleles Amino Acid Sequence Blotting, Northern Blotting, Southern Cell Compartmentation Cloning, Molecular Electrophoresis, Gel, Two-Dimensional Ultrastructure Genes Molecular Sequence Data Morphogenesis Mutation Plant Proteins Immunology Metabolism Restriction Mapping Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Cell Biology |
Content Type | Text |
Resource Type | Article |
Subject | Cell Biology Medicine |
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