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G B Witman

Publications and source records attributed to G B Witman.

At least 19 recordsLinked to original sources
PubMed2005

Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a severe inherited disorder characterised by chronic respiratory disease, male infertility, and, in approximately 50% of affected individuals, a left-right asymmetry defect called situs inversus. PCD is caused by defects in substructures of the ciliary and flagellar axoneme, most commonly loss of the outer dynein arms. Although PCD is believed to involve mutations in many genes, only three have been identified. METHODS: To facilitate discovery of new PCD genes, we have used database searching and analysis to systematically identify the human homologues of proteins associated with the Chlamydomonas reinhardtii outer dynein arm, the best characterised outer arm of any species. RESULTS: We find that 12 out of 14 known Chlamydomonas outer arm subunits have one or more likely orthologues in humans. The results predict a total of 24 human genes likely to encode outer dynein arm subunits and associated proteins possibly necessary for outer arm assembly, plus 12 additional closely related human genes likely to encode inner dynein arm subunits. CONCLUSION: These genes, which have been located on the human chromosomes for easy comparison with known or suspected PCD loci, are excellent candidates for screening for disease-causing mutations in PCD patients with outer and/or inner dynein arm defects.

Animals↗
PubMed2001

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms.

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.

Animals↗
PubMed2001

Differential expression of the C(s) and Calpha1 isoforms of the catalytic subunit of cyclic 3',5'-adenosine monophosphate-dependent protein kinase testicular cells.

The amino terminus of the sperm cAMP-dependent protein kinase catalytic subunit (termed C(s)) differs from that of the Calpha1 isoform expressed in most tissues due to the use of alternative transcripts of the Calpha gene. Both Calpha1 and C(s) transcripts are present in testis; C(s) is expressed specifically in spermatogenic cells and is the only C isoform detected in mature sperm. Immunohistochemistry of mouse testis using antibodies specific for C(s) and Calpha1 now shows that Calpha1 is present in somatic testicular cells, spermatogonia, and preleptotene spermatocytes but not in cells that are in later stages of spermatogenesis. In contrast, C(s) is expressed only in midpachytene and later stage spermatocytes and in spermatids. Therefore, C(s) and Calpha1 expression do not overlap. Immunofluorescence microscopic localization of C(s) in murine and ovine sperm reveals that C(s) is located primarily in sperm tail components, including the midpiece mitochondria and the axoneme. Quantitative analysis of Western blots indicates that individual ovine sperm contain approximately 4 x 10(5) molecules of C(s), a seemingly large number for a protein that acts catalytically.

Animals↗
PubMed2000

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella.

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.

Amino Acid Sequence↗
PubMed2000

Forward and reverse genetic analysis of microtubule motors in Chlamydomonas.

The ability to integrate biochemical, cell biological, and genetic approaches makes Chlamydomonas reinhardtii the premier model organism for studies of the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many that affect dyneins and kinesins. These mutations have yielded much information on the structure and function of the motors as well as the roles of individual subunits within the motors. The development of insertional mutagenesis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with DNA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of insertion, thereby creating mutations that are marked by the integrated DNA. These mutations can be used for forward genetic approaches where one characterizes a mutant phenotype and then clones the relevant gene using the integrated DNA as a tag. The insertional mutants also may be used in a reverse genetic approach in which mutants lacking a gene of interest are identified by DNA hybridization. We describe methods to generate and characterize insertional mutants, using mutations that affect the outer dynein arm as examples.

Animals↗
PubMed2000

The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cells.

cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed C(s), whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine C(s) and Calpha1 (the predominant somatic isoform) now reveal that C(s) is the product of an alternative transcript of the Calpha gene. C(s) cDNA clones from murine and human testes also were isolated and sequenced, indicating that C(s) is of ancient origin and widespread in mammals. In the mouse, C(s) transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that C(s) first appears in pachytene spermatocytes. This is the first demonstration of a cell type-specific expression for any C isoform. The conservation of C(s) throughout mammalian evolution suggests that the unique structure of C(s) is important in the subunit's localization or function within the sperm.

Alternative Splicing↗
PubMed1999

The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly.

Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

Amino Acid Sequence↗
PubMed1999

An insertional mutant of Chlamydomonas reinhardtii with defective microtubule positioning.

cmu1-1 is a new mutation of Chlamydomonas reinhardtii that causes a change in cell shape due to an alteration of cytoplasmic microtubule organization. cmu1 mutant cells were first identified based on their altered cell shape. Unlike wild-type cells, which are ellipsoid, cmu1 cells tend to be either round or egg-shaped with the flagella extending from the narrow end of the cell. Electron microscopic comparison of mutant and wild-type cells indicated that microtubule distribution was altered in the mutant cells. Immunofluorescence microscopy using anti-beta-tubulin antibodies revealed that, in wild-type cells, microtubules arise from the anterior end of the cell in the region of the basal bodies, pass posteriorly subjacent to the plasma membrane, and terminate near the posterior end of the cell. In mutant cells, the microtubules also arise from the basal body region but then become disarrayed. They frequently curl back anteriorly or wrap around the equator of the cell; some microtubules also extend completely to the posterior end of the cell, then turn back toward the anterior end. No changes in the basal body region were detected by electron microscopy. Some cmu1 cells had multiple nuclei or an aberrant number of flagella, both of which may be due to defects in cell division, a process dependent upon microtubules. Thus, cmu1-1, which was generated by insertional mutagenesis and is tagged, appears to encode a protein that plays an essential role in the spatial organization of cytoplasmic microtubules involved in both interphase and mitotic functions.

Animals↗
PubMed1999

LC2, the chlamydomonas homologue of the t complex-encoded protein Tctex2, is essential for outer dynein arm assembly.

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.

Animals↗
PubMed1998

The catalytic subunit of the cAMP-dependent protein kinase of ovine sperm flagella has a unique amino-terminal sequence.

The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated. Ram sperm C was purified and found by mass spectrometry (MS) to be approximately 890 Da smaller than Calpha, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Calpha, but differed from the predicted sequences for the Cbeta and Cgamma isoforms. MS analysis of 2-nitro-5-thiocyanatobenzoic acid fragments showed that the mass difference originated in the amino-terminal region. A unique blocked amino-terminal fragment was isolated from sperm C and sequenced by a combination of tandem mass spectrometry and Edman degradation of a subfragment. The results revealed that the amino-terminal myristate and the first 14 amino acids of Calpha are replaced by an amino-terminal acetate and six different amino acids in sperm C. The predicted mass difference due to these changes is 899 Da. The region of homology between sperm C and Calpha begins at the exon 1/exon 2 boundary in Calpha, suggesting that sperm C results from use of an alternate exon 1 in the Calpha gene. The different amino terminus of sperm C may be related to a unique requirement for localization of the "free" C subunit within the sperm flagellum.

Acetates↗
PubMed1998

A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT).

Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517-1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.

Animals↗
PubMed1997

The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex.

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

Amino Acid Sequence↗
PubMed1997

Functional interaction between Chlamydomonas outer arm dynein subunits: the gamma subunit suppresses the ATPase activity of the alpha beta dimer.

The alpha beta dimer and the gamma subunit of the Chlamydomonas outer arm dynein were solubilized by treating isolated axonemes with 0.6 M KCI, and purified by sucrose density gradient centrifugation. The axonemes were from an ida1 mutant to eliminate contamination of outer arm subunits by inner arm dynein 11, and the axonemes were pre-extracted with 0.6 M CH3COOK to remove non-dynein protein that might otherwise contaminate outer arm dynein fractions in the sucrose gradient. In addition, purer fractions of outer arm dynein subunits were obtained by modifying the centrifugation conditions to take advantage of the propensity of the dynein to dissociate under high hydrostatic pressure in the presence of Mg2+. When sucrose gradient fractions containing the gamma subunit were added to a fraction containing the purified alpha beta dimer under conditions expected to promote reassociation of the subunits to form a trimeric outer arm dynein complex [Takada et al., 1992: J. Biochem, 111:758-762], the total ATPase activity of the mixture was suppressed to a level lower than that of the original alpha beta dimer fraction. The inhibition paralleled the distribution of gamma subunit in the sucrose gradient, was saturable, and was maximum at an approximately equimolar ratio of the gamma subunit to the alpha beta dimer. These results indicate that when the gamma subunit interacts with the alpha beta dimer, the latter's ATPase activity is modulated downward. Previous results showed that interaction of the alpha subunit with the beta subunit suppressed the beta subunit's ATPase activity [Pfister and Witman, 1984: J. Biol. Chem. 259:12072-12080]. Thus, the total ATPase activity of the outer arm dynein is dependent upon communication between all three subunits within the arm.

Adenosine Triphosphatases↗