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Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.

Kato-Minoura T, Hirono M, Kamiya R - J. Cell Biol. (1997)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, School of Science, Nagoya University, Japan.

ABSTRACT
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] = approximately 5.3) but had two novel spots with pIs of approximately 5.6 and approximately 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 wild-type 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.

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Sequence analysis of the ida5 mutation. Sequencing  patterns of the partial cDNA clones obtained from wild type and  ida5 by RT-PCR were shown side by side. (Arrow, right) Position  of a base (C) deletion (asterisks). The altered cDNA and amino  acid sequences are shown at the bottom. The leftmost asparagine  is at amino acid 265. The deletion in ida5 results in a stop codon  TGA at position 268.
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Figure 2: Sequence analysis of the ida5 mutation. Sequencing patterns of the partial cDNA clones obtained from wild type and ida5 by RT-PCR were shown side by side. (Arrow, right) Position of a base (C) deletion (asterisks). The altered cDNA and amino acid sequences are shown at the bottom. The leftmost asparagine is at amino acid 265. The deletion in ida5 results in a stop codon TGA at position 268.

Mentions: The above finding, together with the recent mapping of the actin gene to a locus near that of ida5 (right arm of linkage group XIV) by C. Silflow and P. Lefebvre (personal communication), suggested that the mutant ida5 might have a mutation in the actin-encoding gene itself. We thus analyzed its actin gene. Actin cDNA was isolated from an ida5 cDNA library using the wild-type actin cDNA as the probe. One of the two clones obtained was sequenced over its entire length. The sequence indicated that a base (C) had been deleted from the CCCC sequence starting at nucleotide 838 (in the cDNA nucleotide sequence registered in EMBL/DDBJ/GenBank). The same deletion was found in a cDNA fragment obtained by RTPCR from total ida5 RNA (see Materials and Methods). This deletion should cause a frame shift and produce a stop codon at the amino acid position 268, i.e., at about two-thirds of the total of 377 amino acids (Fig. 2). Hence, no functional actin should be produced in this mutant since the COOH-terminal 110–amino acid sequence is essential to the actin assembly and function (Kabsch et al., 1990). However, although Northern blotting detected the presence of full-length actin mRNA (data not shown), Western blot analysis using an antibody against NH2-terminal 11–amino acid sequence did not detect any truncated actin (see Fig. 4). Truncated products, if any, may be degraded quickly in the cytoplasm.


Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.

Kato-Minoura T, Hirono M, Kamiya R - J. Cell Biol. (1997)

Sequence analysis of the ida5 mutation. Sequencing  patterns of the partial cDNA clones obtained from wild type and  ida5 by RT-PCR were shown side by side. (Arrow, right) Position  of a base (C) deletion (asterisks). The altered cDNA and amino  acid sequences are shown at the bottom. The leftmost asparagine  is at amino acid 265. The deletion in ida5 results in a stop codon  TGA at position 268.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139884&req=5

Figure 2: Sequence analysis of the ida5 mutation. Sequencing patterns of the partial cDNA clones obtained from wild type and ida5 by RT-PCR were shown side by side. (Arrow, right) Position of a base (C) deletion (asterisks). The altered cDNA and amino acid sequences are shown at the bottom. The leftmost asparagine is at amino acid 265. The deletion in ida5 results in a stop codon TGA at position 268.
Mentions: The above finding, together with the recent mapping of the actin gene to a locus near that of ida5 (right arm of linkage group XIV) by C. Silflow and P. Lefebvre (personal communication), suggested that the mutant ida5 might have a mutation in the actin-encoding gene itself. We thus analyzed its actin gene. Actin cDNA was isolated from an ida5 cDNA library using the wild-type actin cDNA as the probe. One of the two clones obtained was sequenced over its entire length. The sequence indicated that a base (C) had been deleted from the CCCC sequence starting at nucleotide 838 (in the cDNA nucleotide sequence registered in EMBL/DDBJ/GenBank). The same deletion was found in a cDNA fragment obtained by RTPCR from total ida5 RNA (see Materials and Methods). This deletion should cause a frame shift and produce a stop codon at the amino acid position 268, i.e., at about two-thirds of the total of 377 amino acids (Fig. 2). Hence, no functional actin should be produced in this mutant since the COOH-terminal 110–amino acid sequence is essential to the actin assembly and function (Kabsch et al., 1990). However, although Northern blotting detected the presence of full-length actin mRNA (data not shown), Western blot analysis using an antibody against NH2-terminal 11–amino acid sequence did not detect any truncated actin (see Fig. 4). Truncated products, if any, may be degraded quickly in the cytoplasm.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, School of Science, Nagoya University, Japan.

ABSTRACT
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] = approximately 5.3) but had two novel spots with pIs of approximately 5.6 and approximately 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 wild-type 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.

Show MeSH
Related in: MedlinePlus