scholarly journals PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella.

1997 ◽  
Vol 8 (3) ◽  
pp. 455-467 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Gene Product ◽  
Insertional Mutagenesis ◽  
Microtubule Assembly ◽  
Flagellar Motility ◽  
Wild Type ◽  
Cellular Functions ◽  
Reading Frame ◽  
Central Apparatus ◽  
Wd Repeats ◽  
Cloned Cdna

The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility.

scholarly journals PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

1996 ◽  
Vol 132 (3) ◽  
pp. 359-370 ◽  
Author(s):  
E F Smith ◽  
P A Lefebvre
Keyword(s):  
Protein Interactions ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Central Pair ◽  
Central Apparatus ◽  
Immunofluorescence Labeling ◽  
Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

scholarly journals PF15p Is the Chlamydomonas Homologue of the Katanin p80 Subunit and Is Required for Assembly of Flagellar Central Microtubules

2004 ◽  
Vol 3 (4) ◽  
pp. 870-879 ◽  
Author(s):  
Erin E. Dymek ◽  
Paul A. Lefebvre ◽  
Elizabeth F. Smith
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Insertional Mutagenesis ◽  
Flagellar Motility ◽  
Wild Type ◽  
Central Pair ◽  
Coding Sequence ◽  
Microtubule Severing ◽  
Central Apparatus

ABSTRACT Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.

scholarly journals The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

1998 ◽  
Vol 9 (12) ◽  
pp. 3351-3365 ◽  
Author(s):  
Catherine A. Perrone ◽  
Pinfen Yang ◽  
Eileen O’Toole ◽  
Winfield S. Sale ◽  
Mary E. Porter
Keyword(s):  
Insertional Mutagenesis ◽  
Gene Transformation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Important Target ◽  
Dynein Intermediate Chain ◽  
Biochemical Analyses ◽  
Intermediate Chain

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm thatida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that theida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

scholarly journals A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest–specific gene product

2003 ◽  
Vol 162 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Gerald Rupp ◽  
Mary E. Porter
Keyword(s):  
Gene Product ◽  
Insertional Mutagenesis ◽  
Growth Arrest ◽  
Coiled Coil ◽  
Specific Gene ◽  
Flagellar Motility ◽  
Wide Range ◽  
Motility Mutant ◽  
Regulatory Complex ◽  
Related Proteins

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.

scholarly journals The Saccharomyces cerevisiae ETH1 Gene, an Inducible Homolog of Exonuclease III That Provides Resistance to DNA-Damaging Agents and Limits Spontaneous Mutagenesis

1999 ◽  
Vol 19 (3) ◽  
pp. 1800-1809 ◽  
Author(s):  
Richard A. O. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Gene Product ◽  
Open Reading Frame ◽  
Endonuclease Activity ◽  
Wild Type ◽  
Ap Endonuclease ◽  
Exonuclease Iii ◽  
Spontaneous Mutagenesis ◽  
Reading Frame

ABSTRACT The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1 + [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were ∼3-fold more sensitive to MMS and ∼10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were ∼15-fold more sensitive to MMS and ∼2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than wereapn1 strains. Elimination of ETH1 inapn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation ofapn1 eth1 cells with an expression vector containingETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in adut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein.

scholarly journals Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

1998 ◽  
Vol 66 (9) ◽  
pp. 4290-4298 ◽  
Author(s):  
Beth A. Bauer ◽  
Marla K. Stevens ◽  
Eric J. Hansen
Keyword(s):  
Gene Product ◽  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Model ◽  
Protein Product ◽  
Haemophilus Ducreyi ◽  
Wild Type ◽  
Reading Frame ◽  
Sexually Transmitted

ABSTRACT The lipooligosaccharide (LOS) present in the outer membrane ofHaemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA(isn) gene of Haemophilus influenzae. InH. influenzae type b, inactivation of the gmhAgene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396–402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficientEscherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a catcartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production byH. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. TheH. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When theH. ducreyi gmhA gene was present in trans ingmhA mutant 35000.252, expression of the gmhAgene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.

scholarly journals Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene from Ralstonia eutropha H16 and Characterization of the Gene Product

2001 ◽  
Vol 183 (1) ◽  
pp. 94-100 ◽  
Author(s):  
Haruhisa Saegusa ◽  
Mari Shiraki ◽  
Chie Kanai ◽  
Terumi Saito
Keyword(s):  
Gene Product ◽  
Ralstonia Eutropha ◽  
Nucleotide Sequence Analysis ◽  
Rich Medium ◽  
Wild Type ◽  
Reading Frame ◽  
Phb Depolymerase ◽  
Freeze Dried ◽  
Dna Fragment

ABSTRACT An intracellular poly[d(−)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric d(−)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutrophawhose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.

scholarly journals Phase Variation in H Type I and Lewis a Epitopes ofHelicobacter pylori Lipopolysaccharide

2000 ◽  
Vol 68 (10) ◽  
pp. 5928-5932 ◽  
Author(s):  
Ben J. Appelmelk ◽  
M. Celeste Martino ◽  
Eveline Veenhof ◽  
Mario A. Monteiro ◽  
Janneke J. Maaskant ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Gene Product ◽  
Insertional Mutagenesis ◽  
Phase Variation ◽  
Type I ◽  
Blood Group Antigens ◽  
Reading Frame ◽  
H Pylori ◽  
Serological Data ◽  
Frequency Phase

ABSTRACT Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Lex), Ley, and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Lex and Ley and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Lex, Ley, H type I, and Lea, yielded a transformant that expressed Lex and Ley. We conclude that JHP563 encodes a β3-galactosyltransferase involved in the biosynthesis of H type I and Lea and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Lex and Lea and had lost both H type I and Leyexpression. Inactivation of HP093-HP094 resulted in a transformant expressing Lex and lacking Ley and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 α2-fucosyltransferase (α2-FucT) gene product is involved in the biosynthesis of both Ley and Lex. Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Lex and Leaexpression, which demonstrates that the HP0379 gene product is both an α3- and an α4-FucT. Our data provide understanding at the molecular level of how H. pylori is able to diversify in the host, a requirement likely essential for successful colonization and transmission.

scholarly journals Regulation of Flagellar Dynein by Calcium and a Role for an Axonemal Calmodulin and Calmodulin-dependent Kinase

2002 ◽  
Vol 13 (9) ◽  
pp. 3303-3313 ◽  
Author(s):  
Elizabeth F. Smith
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Calcium Signal ◽  
Sliding Velocity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Wild Type Level ◽  
Radial Spokes ◽  
Central Apparatus

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonasmutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.

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