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A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product.

Rupp G, Porter ME - J. Cell Biol. (2003)

Bottom Line: The pf2-4 mutant displays an altered waveform that results in slow swimming cells.PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest-specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin.The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.

ABSTRACT
The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest-specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

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Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
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fig1: Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.

Mentions: To identify the gene that was disrupted in 9B11, a fragment of genomic DNA flanking the site of plasmid insertion was obtained by screening a size-fractionated minilibrary with a probe derived from the 3′ end of the NIT1 gene (see Materials and methods; Fig. 1Figure 1.


A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product.

Rupp G, Porter ME - J. Cell Biol. (2003)

Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172716&req=5

fig1: Cloning the PF2 gene. (A) Partial restriction maps of the region containing the PF2 gene from wild-type and pf2-4 (9B11). Also indicated is the location of the NIT1 plasmid insertion in 9B11, now known as pf2-4, and the position of the flanking clone FC-1 (black box) representing the NotI/BamHI restriction fragment recovered from the 9B11 minilibrary. S, SacI sites; N, NotI sites. (B) Southern blot of wild-type (wt) and pf2-4 genomic DNA digested with the indicated restriction enzymes and hybridized with FC-1. (C) Alignment of three overlapping phage clones recovered with FC-1 (black boxes) and two plasmid clones, derived from λG1, that contain the PF2 gene. Plasmid p9-3HA is the epitope-tagged PF2 construct. Also shown are the number of rescued strains obtained by cotransformation of pf2-4 and pf2-1 with the selected clones. nd, not determined.
Mentions: To identify the gene that was disrupted in 9B11, a fragment of genomic DNA flanking the site of plasmid insertion was obtained by screening a size-fractionated minilibrary with a probe derived from the 3′ end of the NIT1 gene (see Materials and methods; Fig. 1Figure 1.

Bottom Line: The pf2-4 mutant displays an altered waveform that results in slow swimming cells.PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest-specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin.The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.

ABSTRACT
The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest-specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

Show MeSH