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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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Electrophoresis patterns of wild-type and mutant  axonemes. (A) Two-dimensional SDS-PAGE pattern of  wild-type axonemes. pH range:  4.0–7.0. (Left) Basic polypeptides. (Bars with numbers)  Positions of molecular mass  standards shown in Mr ×  10−3. (B) Portions of twodimensional electrophoresis  showing spots of actin and  NAP appearing in the mutants. (wt, ida5, and ida5-t)  Axonemes of wild type and  mutants. (wt dynein and ida5  dynein) Inner-arm subspecies g separated by chromatography. Arrows in A and B  indicate the position of actin;  arrowheads indicate those of  NAP. In ida5-t, two spots of  unidentified origins were  shifted by ∼0.2 pH unit to  more alkaline positions (*).  This shift may be caused by  another gene disruption event  in this mutant. The smear  (triangle) is an artifact of silver staining. The faint spots  seen in the dynein patterns (shown in a series) are those of tubulins with various degrees of posttranslational modification. (C) One- dimensional SDS-PAGE of subspecies g. Samples from wild type, ida5, and their mixture were loaded on the same 11% polyacrylamide  gel. (Lane rabbit skeletal) Rabbit skeletal muscle actin. Only a portion near the actin band is shown. All gels were stained with silver.
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Figure 1: Electrophoresis patterns of wild-type and mutant axonemes. (A) Two-dimensional SDS-PAGE pattern of wild-type axonemes. pH range: 4.0–7.0. (Left) Basic polypeptides. (Bars with numbers) Positions of molecular mass standards shown in Mr × 10−3. (B) Portions of twodimensional electrophoresis showing spots of actin and NAP appearing in the mutants. (wt, ida5, and ida5-t) Axonemes of wild type and mutants. (wt dynein and ida5 dynein) Inner-arm subspecies g separated by chromatography. Arrows in A and B indicate the position of actin; arrowheads indicate those of NAP. In ida5-t, two spots of unidentified origins were shifted by ∼0.2 pH unit to more alkaline positions (*). This shift may be caused by another gene disruption event in this mutant. The smear (triangle) is an artifact of silver staining. The faint spots seen in the dynein patterns (shown in a series) are those of tubulins with various degrees of posttranslational modification. (C) One- dimensional SDS-PAGE of subspecies g. Samples from wild type, ida5, and their mixture were loaded on the same 11% polyacrylamide gel. (Lane rabbit skeletal) Rabbit skeletal muscle actin. Only a portion near the actin band is shown. All gels were stained with silver.

Mentions: Fractionation by chromatography of the high salt extract of ida5 axonemes showed that its axonemes lack four (a, c, d, and e) of the seven (a–g) inner-arm dynein subspecies normally present in the wild-type axoneme (Kato et al., 1993). SDS-PAGE patterns of the dynein fractions indicated that its subspecies b and g had a 43-kD band at a position similar to that of the actin band in the wild-type counterparts (Kato et al., 1993). However, two-dimensional gel electrophoresis of the axoneme revealed a striking difference (Fig. 1, A and B); the ida5 axoneme lacked the spot corresponding to that of actin (isoelectric point [pI] = ∼5.3) but had two novel spots at apparent pIs of ∼5.6 and ∼5.7. The same change in the actin spot was observed with the dynein fractions b (data not shown) and g (Fig. 1 B). In one-dimensional SDS-PAGE, the 43-kD band in ida5 had a slightly larger mobility than that in wild type (Fig. 1 C).


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)

Electrophoresis patterns of wild-type and mutant  axonemes. (A) Two-dimensional SDS-PAGE pattern of  wild-type axonemes. pH range:  4.0–7.0. (Left) Basic polypeptides. (Bars with numbers)  Positions of molecular mass  standards shown in Mr ×  10−3. (B) Portions of twodimensional electrophoresis  showing spots of actin and  NAP appearing in the mutants. (wt, ida5, and ida5-t)  Axonemes of wild type and  mutants. (wt dynein and ida5  dynein) Inner-arm subspecies g separated by chromatography. Arrows in A and B  indicate the position of actin;  arrowheads indicate those of  NAP. In ida5-t, two spots of  unidentified origins were  shifted by ∼0.2 pH unit to  more alkaline positions (*).  This shift may be caused by  another gene disruption event  in this mutant. The smear  (triangle) is an artifact of silver staining. The faint spots  seen in the dynein patterns (shown in a series) are those of tubulins with various degrees of posttranslational modification. (C) One- dimensional SDS-PAGE of subspecies g. Samples from wild type, ida5, and their mixture were loaded on the same 11% polyacrylamide  gel. (Lane rabbit skeletal) Rabbit skeletal muscle actin. Only a portion near the actin band is shown. All gels were stained with silver.
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Figure 1: Electrophoresis patterns of wild-type and mutant axonemes. (A) Two-dimensional SDS-PAGE pattern of wild-type axonemes. pH range: 4.0–7.0. (Left) Basic polypeptides. (Bars with numbers) Positions of molecular mass standards shown in Mr × 10−3. (B) Portions of twodimensional electrophoresis showing spots of actin and NAP appearing in the mutants. (wt, ida5, and ida5-t) Axonemes of wild type and mutants. (wt dynein and ida5 dynein) Inner-arm subspecies g separated by chromatography. Arrows in A and B indicate the position of actin; arrowheads indicate those of NAP. In ida5-t, two spots of unidentified origins were shifted by ∼0.2 pH unit to more alkaline positions (*). This shift may be caused by another gene disruption event in this mutant. The smear (triangle) is an artifact of silver staining. The faint spots seen in the dynein patterns (shown in a series) are those of tubulins with various degrees of posttranslational modification. (C) One- dimensional SDS-PAGE of subspecies g. Samples from wild type, ida5, and their mixture were loaded on the same 11% polyacrylamide gel. (Lane rabbit skeletal) Rabbit skeletal muscle actin. Only a portion near the actin band is shown. All gels were stained with silver.
Mentions: Fractionation by chromatography of the high salt extract of ida5 axonemes showed that its axonemes lack four (a, c, d, and e) of the seven (a–g) inner-arm dynein subspecies normally present in the wild-type axoneme (Kato et al., 1993). SDS-PAGE patterns of the dynein fractions indicated that its subspecies b and g had a 43-kD band at a position similar to that of the actin band in the wild-type counterparts (Kato et al., 1993). However, two-dimensional gel electrophoresis of the axoneme revealed a striking difference (Fig. 1, A and B); the ida5 axoneme lacked the spot corresponding to that of actin (isoelectric point [pI] = ∼5.3) but had two novel spots at apparent pIs of ∼5.6 and ∼5.7. The same change in the actin spot was observed with the dynein fractions b (data not shown) and g (Fig. 1 B). In one-dimensional SDS-PAGE, the 43-kD band in ida5 had a slightly larger mobility than that in wild type (Fig. 1 C).

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