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© The Rockefeller University Press,
0021-9525/1997//649 $5.00
The Journal of Cell Biology, Volume 137, Number 3,
, 1997 649-656
Article |
Chlamydomonas Inner-Arm Dynein Mutant, ida5, Has a Mutation in an Actin-encoding Gene
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Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; and National Institute for Basic Biology, Okazaki 444, Japan
Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5' end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] =
5.3) but had two novel spots with pIs of 5.6 and 5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wildtype cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.
Abbreviations used in this paper: IBMX, 3-isobutyl-1-methylxanthine; mt+, mating type plus; mt–, mating type minus; NAP, novel actin-like protein; RT, reverse transcription; TAP, Tris-acetic acid-phosphate.
Please address all correspondence to Ritsu Kamiya, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, 113 Tokyo, Japan. Tel. and Fax: (81) 3-5800-6842. e-mail: kamiyar{at}biol.s.u-tokyo.ac.jp
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