RESUMO
The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Ciliopatias/genética , Anormalidades Craniofaciais/genética , Nanismo/genética , Orelha/anormalidades , Pescoço/anormalidades , Síndromes Orofaciodigitais/genética , Tórax/anormalidades , Adolescente , Adulto , Ciliopatias/fisiopatologia , Consanguinidade , Anormalidades Craniofaciais/fisiopatologia , Nanismo/fisiopatologia , Orelha/fisiopatologia , Síndrome de Ellis-Van Creveld/genética , Síndrome de Ellis-Van Creveld/fisiopatologia , Exoma/genética , Feminino , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Mutação , Pescoço/fisiopatologia , Síndromes Orofaciodigitais/fisiopatologia , Fenótipo , Tórax/fisiopatologia , Adulto JovemRESUMO
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.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Dineínas/metabolismo , Flagelos/fisiologia , Microtúbulos/fisiologia , Animais , Divisão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestrutura , Citoplasma/fisiologia , Dineínas/biossíntese , Flagelos/ultraestrutura , Deleção de Genes , Genes de Plantas , Microscopia Eletrônica , Microscopia de Vídeo , Microtúbulos/ultraestrutura , Movimento , MutagêneseRESUMO
Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Rodopsina/fisiologia , Transdução de Sinais , Animais , DNA/fisiologia , Análise Mutacional de DNA , Eletrofisiologia , Luz , Mutagênese Insercional , Rodopsina/genéticaRESUMO
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.
Assuntos
Dineínas/química , Dineínas/metabolismo , Flagelos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Clonagem Molecular , Citoplasma/metabolismo , Dineínas do Citoplasma , Dineínas/genética , Flagelos/ultraestrutura , Deleção de Genes , Genes de Protozoários , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Proteínas Motores Moleculares , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Homologia de Sequência de AminoácidosRESUMO
We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Dineínas/química , Dineínas/metabolismo , Flagelos/fisiologia , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestrutura , Sequência Consenso , Dineínas/genética , Flagelos/ultraestrutura , Substâncias Macromoleculares , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Sondas de Oligonucleotídeos , Biossíntese de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
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.
Assuntos
Chlamydomonas reinhardtii/genética , Dineínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii/química , Clonagem Molecular , DNA Complementar/isolamento & purificação , DNA de Plantas/isolamento & purificação , Dineínas/química , Flagelos/química , Flagelos/ultraestrutura , Genes de Plantas/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional/fisiologia , Análise de Sequência de DNARESUMO
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.
Assuntos
Chlamydomonas/genética , Cílios/metabolismo , Flagelos/metabolismo , Rim Policístico Autossômico Recessivo/genética , Proteínas/química , Proteínas de Protozoários/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Chlamydomonas/citologia , Cílios/genética , Cílios/patologia , Cílios/ultraestrutura , Clonagem Molecular , Sequência Conservada , Flagelos/genética , Flagelos/patologia , Flagelos/ultraestrutura , Humanos , Rim/metabolismo , Rim/patologia , Meiose , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/patologia , Proteínas Motores Moleculares/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Proteínas de Plantas , Rim Policístico Autossômico Recessivo/patologia , Rim Policístico Autossômico Recessivo/fisiopatologia , Ligação Proteica , Subunidades Proteicas , Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Dineínas/genética , Síndrome de Kartagener/genética , Animais , Chlamydomonas/genética , Cílios/genética , Genoma Humano/genética , Humanos , Filogenia , Isoformas de ProteínasRESUMO
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.
Assuntos
Chlamydomonas reinhardtii/genética , Dineínas/genética , Genes de Protozoários , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos , Proteínas de Protozoários/genética , Animais , Movimento Celular , Clonagem Molecular , Flagelos/genética , Flagelos/ultraestrutura , Camundongos , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Fenótipo , Proteínas de Protozoários/metabolismo , Homologia de Sequência , Transformação Genética , Ubiquitina-Proteína Ligases , Região do Complexo-t do GenomaRESUMO
Inturned (INTU), a cilia and planar polarity effector, performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for intraflagellar transport A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily responds to the Hh pathway activation. Depleting Intu blocked Smo agonist-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.
Assuntos
Carcinoma Basocelular/metabolismo , Cílios/metabolismo , Cílios/patologia , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/genética , Neoplasias Cutâneas/metabolismo , Animais , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas Hedgehog/genética , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , TransfecçãoRESUMO
High-voltage electroporation was used to transform Agrobacterium tumefaciens strains A136 and A348, reaching the efficiency of 1-3 x 10(8) transformants/micrograms DNA. Transformation frequency was dependent on the electrical field strength and the pulse length. No significant reduction in transformation efficiency was observed when the transforming DNA contained sites sensitive to endonuclease AtuCI of A. tumefaciens.
Assuntos
Rhizobium/genética , Transformação Genética , Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletricidade , Vetores Genéticos , PlasmídeosRESUMO
A virB-lacZ translational fusion was constructed to monitor expression of the Agrobacterium tumefaciens virB operon. Expression of the fusion gene was dependent on the presence of pTiA6 virA, virG, and a plant factor acetosyringone. Analysis of deletion mutants, constructed by exonuclease Bal31 digestion, showed that 68 residues upstream of the virB transcription initiation site was necessary for its expression. A TT----CC substitution at positions -62 and -61 led to a 7 fold reduction in virB expression. The virB upstream region contains a tetradecameric sequence, dPuT/ATDCAATGHAAPy (D = A, G or T; H = A, C or T), that is conserved in the non-transcribed regions of all vir genes. Alteration of the position of this sequence relative to the promoter region sequences had a drastic negative effect on virB expression.
Assuntos
Genes Bacterianos , Óperon , Sequências Reguladoras de Ácido Nucleico , Rhizobium/genética , Sequência de Bases , Dados de Sequência Molecular , Testes de Mutagenicidade , Plasmídeos , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Chlamydomonas/genética , Microtúbulos/genética , Proteínas Motores Moleculares/genética , Mutagênese Insercional/métodos , Animais , Dineínas/genética , MutaçãoRESUMO
The Agrobacterium tumefaciens Ti plasmid virG locus, in conjunction with virA and acetosyringone, activates transcription of the virulence (vir) genes. Insertional and deoxyoligonucleotide-directed mutagenesis studies showed that both octopine and nopaline Ti plasmid virG genes initiate translation at a UUG codon. VirG protein initiated at this UUG codon was found to be 241 amino acid residues in length and had an apparent molecular mass of 27.1 kilodaltons. A Salmonella typhimurium trp-virG transcriptional fusion was constructed to overproduce VirG. Agrobacterium cells containing this gene fusion showed a large increase in virG activity in the presence of virA and acetosyringone. Since the trp promoter is not under virA-virG control, this result indicates that modification of VirG is necessary for its full activity. VirG overproduced in Escherichia coli was purified from inclusion bodies. It was found to be a DNA-binding protein that preferentially bound DNA fragments containing the 5' nontranscribed regions of the virA, -B, -C, -D, and -G operons. Significant specific binding to the 5' nontranscribed region sequences of virE was not detected. DNase I footprinting of the upstream regions of virC-virD and virG showed that VirG binds to sequences around the vir box region.
Assuntos
Proteínas de Bactérias/metabolismo , Códon/genética , Proteínas de Ligação a DNA/metabolismo , Genes Bacterianos , Biossíntese de Proteínas , RNA Mensageiro/genética , Rhizobium/genética , Fatores de Transcrição , Ativação Transcricional , Sequência de Bases , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos , Rhizobium/metabolismo , Rhizobium/patogenicidade , VirulênciaRESUMO
Expression of Agrobacterium tumefaciens virulence (vir) genes is dependent on the presence of a conserved 'vir box' sequence in their 5' nontranscribed regions. The location and number of these sequences vary considerably in different vir genes. Site-directed mutagenesis was used to identify the functional vir box(es) of virB, virC and virD. For virB expression both vir box B1 and B2 are required but only the vir box B1 is absolutely essential. Of the five vir boxes of virC and virD two are required for virC expression while only one vir box is required for virD expression. To investigate the minimum sequences necessary for vir gene induction a deletion derivative of virE that lacks the vir box region was used. This mutant is not induced by acetosyringone. The inducibility of this promoter was restored when a synthetic deoxyoligonucleotide dGTTTCAATTGAAAC was introduced at a location analogous to that of the wild type vir box sequence. Mutational analysis indicate that the functional vir box sequence is 14 residues in length, contains a dyad symmetry and has the consensus sequence d ryTncAaTTGnAaY [corrected] (r = purine, y = pyrimidine).
Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Rhizobium/genética , Sequência de Bases , Sítios de Ligação , Deleção Cromossômica , Cromossomos Bacterianos , Sequência Consenso , Análise Mutacional de DNA , DNA Bacteriano/genética , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Ativação TranscricionalRESUMO
The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.
Assuntos
Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Canais Iônicos , Rhizobium/metabolismo , Desoxirribonucleoproteínas/ultraestrutura , Microscopia Eletrônica , Peso Molecular , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Relação Estrutura-AtividadeRESUMO
The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica/genética , Fatores de Transcrição , Fatores de Virulência , Virulência/genética , Acetofenonas/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.
Assuntos
Genes Bacterianos , Mutagênese , Rhizobium/genética , Sequência de Aminoácidos , Códon/genética , Escherichia coli/genética , Expressão Gênica , Genótipo , Dados de Sequência Molecular , Nitrosoguanidinas/farmacologia , Fenótipo , Plasmídeos , Proteínas Recombinantes/metabolismo , Rhizobium/efeitos dos fármacos , Rhizobium/patogenicidade , Rhizobium/efeitos da radiação , Raios Ultravioleta , Virulência/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
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.