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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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Southern hybridization analyses at different stringencies. Probe 2 in Fig. 3 was used. n, restriction fragments from nit1/ cw15 (parent strain); t, fragments from ida5-t. For abbreviations  of restriction enzymes, see Fig. 3 legend. Experimental procedure  was the same as in Fig. 3 A except for the temperature at which  hybridization and wash were performed. At lower stringencies,  an additional band (*) appears in each lane of both samples.
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Figure 8: Southern hybridization analyses at different stringencies. Probe 2 in Fig. 3 was used. n, restriction fragments from nit1/ cw15 (parent strain); t, fragments from ida5-t. For abbreviations of restriction enzymes, see Fig. 3 legend. Experimental procedure was the same as in Fig. 3 A except for the temperature at which hybridization and wash were performed. At lower stringencies, an additional band (*) appears in each lane of both samples.

Mentions: As stated, our previous study indicated that Chlamydomonas has only a single copy of actin-encoding gene (Sugase et al., 1996). However, the above finding that the mutants ida5 and ida5-t with serious deletions in the actin gene grow normally has raised the possibility that another protein(s), such as NAP, substitutes for actin in important functions in these mutants, because actin has been believed to be essential for cellular function. The substituting protein, if any, may be structurally related to actin. Thus, we examined if Chlamydomonas has other genes that become hybridized with a fragment of the conventional actin gene at low stringency. Fig. 8 shows the results of such an experiment. Under the conditions shown in Fig. 3, probe 2 did not hybridize with any band in ida5-t, whereas in the parent (nit1/cw15) it hybridized with one or two bands, at positions exactly as expected from the restriction map of the known actin gene. When the stringency was slightly decreased by lowering the temperature from 67° to 65°C, an additional band appeared in each sample of the DNA fragments of both strains. This band cannot be explained from the sequence of the conventional actin gene. With additional lowering of stringency, it was obscured by many other nonspecific bands.


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)

Southern hybridization analyses at different stringencies. Probe 2 in Fig. 3 was used. n, restriction fragments from nit1/ cw15 (parent strain); t, fragments from ida5-t. For abbreviations  of restriction enzymes, see Fig. 3 legend. Experimental procedure  was the same as in Fig. 3 A except for the temperature at which  hybridization and wash were performed. At lower stringencies,  an additional band (*) appears in each lane of both samples.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Southern hybridization analyses at different stringencies. Probe 2 in Fig. 3 was used. n, restriction fragments from nit1/ cw15 (parent strain); t, fragments from ida5-t. For abbreviations of restriction enzymes, see Fig. 3 legend. Experimental procedure was the same as in Fig. 3 A except for the temperature at which hybridization and wash were performed. At lower stringencies, an additional band (*) appears in each lane of both samples.
Mentions: As stated, our previous study indicated that Chlamydomonas has only a single copy of actin-encoding gene (Sugase et al., 1996). However, the above finding that the mutants ida5 and ida5-t with serious deletions in the actin gene grow normally has raised the possibility that another protein(s), such as NAP, substitutes for actin in important functions in these mutants, because actin has been believed to be essential for cellular function. The substituting protein, if any, may be structurally related to actin. Thus, we examined if Chlamydomonas has other genes that become hybridized with a fragment of the conventional actin gene at low stringency. Fig. 8 shows the results of such an experiment. Under the conditions shown in Fig. 3, probe 2 did not hybridize with any band in ida5-t, whereas in the parent (nit1/cw15) it hybridized with one or two bands, at positions exactly as expected from the restriction map of the known actin gene. When the stringency was slightly decreased by lowering the temperature from 67° to 65°C, an additional band appeared in each sample of the DNA fragments of both strains. This band cannot be explained from the sequence of the conventional actin gene. With additional lowering of stringency, it was obscured by many other nonspecific bands.

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