Limits...
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.

Show MeSH

Related in: MedlinePlus

Southern (A) and  Northern (B) hybridization  analyses of ida5-t. Along with  the actin gene, tubulin gene  and message were analyzed  as controls. (A) Genomic  DNAs from nit1/cw15 (n)  and ida5-t (t) were digested  with a restriction enzyme  shown at the bottom and  probed with the probes indicated in the restriction map  (Sugase et al., 1996). The  solid and open boxes indicate  coding exons and transcribed  UTR. Pv, PvuII; ScII, SacII;  Ps, PstI; Bg, BglII; ScI, SacI;  Sal, SalI; E, EcoRI; X, XhoI;  H, HindIII. The membranes  were hybridized at 67°C  overnight. (Bars with numbers) Positions of the λHindIII markers shown in bp ×  10−3. (B) Total RNA was  probed with either wild-type  actin cDNA (act) or TubA1  fragment (tub). Hybridization was performed at 65°C.  (Arrow) Bands of actin and  α-tubulin mRNAs (almost  the same size).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139884&req=5

Figure 3: Southern (A) and Northern (B) hybridization analyses of ida5-t. Along with the actin gene, tubulin gene and message were analyzed as controls. (A) Genomic DNAs from nit1/cw15 (n) and ida5-t (t) were digested with a restriction enzyme shown at the bottom and probed with the probes indicated in the restriction map (Sugase et al., 1996). The solid and open boxes indicate coding exons and transcribed UTR. Pv, PvuII; ScII, SacII; Ps, PstI; Bg, BglII; ScI, SacI; Sal, SalI; E, EcoRI; X, XhoI; H, HindIII. The membranes were hybridized at 67°C overnight. (Bars with numbers) Positions of the λHindIII markers shown in bp × 10−3. (B) Total RNA was probed with either wild-type actin cDNA (act) or TubA1 fragment (tub). Hybridization was performed at 65°C. (Arrow) Bands of actin and α-tubulin mRNAs (almost the same size).

Mentions: Unexpectedly, Southern blot analyses of the ida5-t genome using four different fragments from the wild-type actin genomic clone as probes did not detect bands that hybridized with any of the probes. With the parent strain nit1/cw15, on the other hand, the same four probes clearly detected bands at positions exactly as predicted from the sequence (Fig. 3 A). These findings indicate that a large portion of the actin-encoding region is missing from ida5-t. Such loss of a gene upon insertional mutagenesis has often been observed before, although its exact mechanism is unknown (Tam and Lefebvre, 1993).


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 (A) and  Northern (B) hybridization  analyses of ida5-t. Along with  the actin gene, tubulin gene  and message were analyzed  as controls. (A) Genomic  DNAs from nit1/cw15 (n)  and ida5-t (t) were digested  with a restriction enzyme  shown at the bottom and  probed with the probes indicated in the restriction map  (Sugase et al., 1996). The  solid and open boxes indicate  coding exons and transcribed  UTR. Pv, PvuII; ScII, SacII;  Ps, PstI; Bg, BglII; ScI, SacI;  Sal, SalI; E, EcoRI; X, XhoI;  H, HindIII. The membranes  were hybridized at 67°C  overnight. (Bars with numbers) Positions of the λHindIII markers shown in bp ×  10−3. (B) Total RNA was  probed with either wild-type  actin cDNA (act) or TubA1  fragment (tub). Hybridization was performed at 65°C.  (Arrow) Bands of actin and  α-tubulin mRNAs (almost  the same size).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Southern (A) and Northern (B) hybridization analyses of ida5-t. Along with the actin gene, tubulin gene and message were analyzed as controls. (A) Genomic DNAs from nit1/cw15 (n) and ida5-t (t) were digested with a restriction enzyme shown at the bottom and probed with the probes indicated in the restriction map (Sugase et al., 1996). The solid and open boxes indicate coding exons and transcribed UTR. Pv, PvuII; ScII, SacII; Ps, PstI; Bg, BglII; ScI, SacI; Sal, SalI; E, EcoRI; X, XhoI; H, HindIII. The membranes were hybridized at 67°C overnight. (Bars with numbers) Positions of the λHindIII markers shown in bp × 10−3. (B) Total RNA was probed with either wild-type actin cDNA (act) or TubA1 fragment (tub). Hybridization was performed at 65°C. (Arrow) Bands of actin and α-tubulin mRNAs (almost the same size).
Mentions: Unexpectedly, Southern blot analyses of the ida5-t genome using four different fragments from the wild-type actin genomic clone as probes did not detect bands that hybridized with any of the probes. With the parent strain nit1/cw15, on the other hand, the same four probes clearly detected bands at positions exactly as predicted from the sequence (Fig. 3 A). These findings indicate that a large portion of the actin-encoding region is missing from ida5-t. Such loss of a gene upon insertional mutagenesis has often been observed before, although its exact mechanism is unknown (Tam and Lefebvre, 1993).

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