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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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Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
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fig2: Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.

Mentions: Selected restriction fragments from the phage clones were used to probe Southern and Northern blots to define the boundaries of the PF2 gene. Genomic Southern blots probed with subclones A–D indicated that the NIT1 plasmid inserted into a 3.5-kb SacI restriction fragment without significant deletion of the surrounding genomic DNA in pf2-4 (Fig. 2Figure 2.


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)

Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Analysis of the PF2 transcription unit. (A) Partial restriction map of wild-type genomic DNA in the region of the PF2 transcription unit. Also shown are selected SacI fragments (A, C, and D) and a NotI fragment (B) that were used to map the boundaries of the plasmid insertion and determine the size of the transcription unit. (B) Northern blot loaded with total RNA isolated from wild-type (wt) and pf2 mutant cells before (0) and 45 min after deflagellation. This blot was hybridized with probe C, which recognized an ∼2.5-kb transcript that is up-regulated in wild-type cells and missing in pf2 mutants. (B, bottom) Loading control. The blot was rehybridized with a CRY1 probe, which encodes the ribosomal S14 protein subunit (Nelson et al., 1994). (C) Diagram of the intron–exon structure of the PF2 transcription unit. Open rectangles indicate exons and solid lines indicate introns. The predicted translation start (ATG) site and the putative polyadenylation signal (Poly A) are indicated. Also shown are the single nucleotide alterations that resulted in the pf2-1 and pf2-2 mutations, and the location of the epitope tag in exon 12.
Mentions: Selected restriction fragments from the phage clones were used to probe Southern and Northern blots to define the boundaries of the PF2 gene. Genomic Southern blots probed with subclones A–D indicated that the NIT1 plasmid inserted into a 3.5-kb SacI restriction fragment without significant deletion of the surrounding genomic DNA in pf2-4 (Fig. 2Figure 2.

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