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1.
Proc Biol Sci ; 265(1399): 869-74, 1998 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-9633112

RESUMO

The rod photoreceptors of the European eel, Anguilla anguilla (L.), alter their wavelength of maximum sensitivity (lambda max) from c.a. 523 nm to c.a. 482 nm at maturation, a switch involving the synthesis of a new visual pigment protein (opsin) that is inserted into the outer segments of existing rods. We artificially induced the switch in rod opsin production by the administration of hormones, and monitored the switch at the level of mRNA accumulation using radiolabelled oligonuleotides that hybridized differently to the two forms of eel rod opsin. The production of the deep-sea form of rod opsin was detected 6 h after the first hormone injection, and the switch in rod opsin expression was complete within four weeks, at which time only the mRNA for the deep-sea opsin was detectable in the retinal cells. It is suggested that this system could be used as a tractable model for studying the regulatory control of opsin gene expression.


Assuntos
Anguilla/metabolismo , Regulação da Expressão Gênica , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Opsinas de Bastonetes/genética , Animais , Gonadotropina Coriônica , Masculino , Microespectrofotometria , Hormônios Hipofisários , Pigmentos da Retina , Testosterona/sangue
2.
DNA Seq ; 7(2): 97-106, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9063646

RESUMO

Only two gas vesicle genes have been previously identified in the cyanobacteria, gvpA and gvpC, both of which encode structural gas vesicle proteins. Analysis of the nucleotide sequence immediately downstream of gvpC in the cyanobacterium Anabaena flos-aquae has revealed the presence of 4 ORFs (open reading frames) the products of which share significant homology with a number of the gene products derived from halobacterial gvp gene clusters. In halobacteria the gas vesicle gene clusters consist of 14 genes involved in gas vesicle synthesis and assembly. The product of Anabaena ORF 1, located immediately downstream of gvpC is homologous to halobacterial GvpNs. For the remaining ORFs the predicted gene products show homology to both GvpJ and GvpA for ORF 2, to GvpK and GvpA for ORF 3, and to both GvpF and GvpL for ORF 4.


Assuntos
Anabaena/genética , Proteínas de Bactérias , Genes Bacterianos , Halobacterium/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Homologia de Sequência
4.
Arch Microbiol ; 164(1): 50-7, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7544103

RESUMO

Southern analysis of genomic DNA from Anabaena flos-aquae revealed that the genes encoding the two authenticated protein components of cyanobacterial gas vesicles, GvpA and GvpC, were carried on the same 4.9-kb HindIII restriction fragment. By comparing the hybridization intensities observed when either gvpA- or gvpC-specific oligonucleotides are bound to this HindIII fragment, we calculated that the A. flos-aquae genome contains seven copies of gvpA and a single copy of gvpC. The nucleotide sequence of the longest cloned section of the gvpA/C cluster of A. flos-aquae DNA revealed the presence of four complete copies of gvpA and part of a fifth copy located upstream from a single copy of gvpC; no clones carrying the entire gvpA/C-bearing HindIII fragment were identified. The distribution of Sau3A restriction sites throughout the gvpA/C-bearing genomic HindIII fragment resembled that seen in the cloned portion of the gvpA/C cluster and is consistent with that expected for a cluster containing seven copies of gvpA and one copy of gvpC. The length of transcripts that hybridize to both gvpA and gvpC on Northern blots was consistent with a 7gvpA + 1gvpC transcriptional unit.


Assuntos
Anabaena/genética , Proteínas de Bactérias/genética , Dosagem de Genes , Genes Bacterianos/genética , Proteínas de Membrana/genética , Família Multigênica/genética , Proteínas , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Mapeamento por Restrição , Análise de Sequência de DNA
5.
Biochem J ; 236(1): 31-6, 1986 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-3098234

RESUMO

Gas vesicles of cyanobacteria are formed by a protein called 'gas-vesicle protein' (GVP). The complete amino acid sequence has been determined of GVP from Anabaena flos-aquae. It is 70 residues long and has an Mr of 7388. This corresponds to the size of the repeating unit cell demonstrated by X-ray crystallography of intact gas vesicles. Details of the sequence are related to the secondary beta-sheet structure of the protein and its contrasting hydrophilic and hydrophobic surfaces. Extensive amino acid sequences have also been determined for GVPs from two other cyanobacteria, species of Calothrix and Microcystis; they are highly homologous with that of Anabaena GVP. Electrophoretic analysis indicates that GVPs of different cyanobacteria form a variety of stable oligomers.


Assuntos
Proteínas de Bactérias , Cianobactérias/análise , Proteínas de Plantas , Proteínas , Sequência de Aminoácidos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Fragmentos de Peptídeos/análise , Difração de Raios X
6.
Arch Microbiol ; 157(3): 229-34, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1510555

RESUMO

The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea. Recombinant GvpC was purified from inclusion bodies, produced in an E. coli strain containing an expression vector bearing the gene encoding GvpC from A. flos-aquae, and then solubilised in 6 M urea. This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present. The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value. These results indicate that the function of GvpC is to increase the strength of the structure.


Assuntos
Anabaena/genética , Proteínas de Bactérias , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Vacúolos/química , Anabaena/metabolismo , Extratos Celulares , Clonagem Molecular , Gases/metabolismo , Pressão Hidrostática , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Ureia
7.
J Gen Microbiol ; 138(6): 1243-50, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1527496

RESUMO

In addition to GvpA, the main structural protein, an SDS-soluble protein has been found in gas vesicles isolated from six different genera of cyanobacteria. N-terminal sequence analysis of the first 30 to 60 residues of the gel-purified proteins showed that they were homologous to GvpC, a protein that strengthens the gas vesicle in Anabaena flos-aquae. The proteins from some of the organisms showed rather low homology, however, and this may explain why the genes that encode them have not been found by Southern hybridization studies. The gas vesicles of another cyanobacterium, Dactylococcopsis salina, contained two SDS-soluble proteins (M(r) 17,000 and 35,000) that were identical in sequence for the first 24 residues but not thereafter; these two proteins showed no clear homology to GvpC. The sequence of GvpA, the main structural gas vesicle protein, was very similar in each of the organisms investigated. GvpA from the purple bacterium Amoebobacter pendens was different for the first 8 residues but 51 of the next 56 residues were identical to those of the cyanobacterial GvpA. Analysis of the GvpA and GvpC sequences provides support for the idea that the low diversity of GvpA reflects a high degree of conservation rather than a recent origin followed by lateral gene transfer between different bacteria.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Cianobactérias/química , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
8.
Mol Microbiol ; 17(1): 147-54, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7476201

RESUMO

We have previously shown that the gas-vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33-RR) sandwiched between 18 N-terminal and 10 C-terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33-amino-acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33-RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33-amino-acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.


Assuntos
Anabaena/fisiologia , Proteínas de Membrana/metabolismo , Proteínas , Vacúolos/química , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Sequência de Bases , Sequência Conservada , Gases , Corpos de Inclusão/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Dados de Sequência Molecular , Pressão , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/metabolismo
9.
J Gen Microbiol ; 139(10): 2353-63, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8254305

RESUMO

Previous studies have shown that gas vesicles isolated from the cyanobacterium Anabaena flos-aquae contain two types of protein, GvpA, a small hydrophobic protein that forms the main ribbed structure, and GvpC, a protein comprising five repeats of a 33-amino-acid-residue motif, which is located on the outer surface of the GvpA shell. GvpC was shown to increase the critical collapse pressure of the gas vesicles; it was thought to do this by forming a series of molecular ties that bind the ribs together. We now show that antibodies raised against GvpC label both the central cylinders and the conical end caps of native gas vesicles but fail to bind to gas vesicles that have been stripped of GvpC. The molar ratio of GvpA to GvpC has been calculated from amino acid analyses of gas vesicle hydrolysates by reference to the abundance of amino acids that occur predominantly or exclusively in one protein or the other; the molar ratio was found to be 25:1 in freshly isolated gas vesicles and 23:1 in gas vesicles saturated with GvpC. We have considered three ways in which the 33-residue repeats of GvpC might interact with the crystallographic unit cell of GvpA molecules in the ribs. The Anabaena GvpC will bind to and restore the strength of gas vesicles isolated from Aphanizomenon and Microcystis that lack their native GvpC.


Assuntos
Anabaena/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas , Vacúolos/química , Aminoácidos/análise , Anabaena/ultraestrutura , Anticorpos Antibacterianos/análise , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/imunologia , Fracionamento Celular , Cianobactérias/química , Cianobactérias/ultraestrutura , Gases , Proteínas de Membrana/imunologia , Pressão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Vacúolos/ultraestrutura
10.
J Gen Microbiol ; 137(10): 2395-400, 1991 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1770354

RESUMO

Antibodies were raised against intact gas vesicles of Anabaena flos-aquae, and against a synthetic peptide (GVPaNT) whose sequence is identical to the N-terminal region of the main gas vesicle protein, GVPa. A two-stage centrifugation procedure is described for separating gold-labelled antibodies bound to gas vesicles from unbound antibodies. The GVPaNT antibody bound to gas vesicles that had been previously rinsed with SDS to remove the outer gas vesicle protein, GVPc. Treatment with this antibody caused the gas vesicles to aggregate together end-to-end rather than side-by-side. The binding of the anti-GVPaNT-immunogold particles to the gas vesicle was restricted to the conical ends of the structure. These observations indicate that the sequence to which the GVPaNT antibodies were raised, residues 1 to 13 of the GVPa molecule, is exposed only at the outer surface of the cones and that it is normally obscured by GVPc. As GVPa forms both the conical ends and the cylindrical midsection of the gas vesicle, exposure of the N-terminal sequence only in the cones must be due to differences in the contact between adjacent GVPa molecules in the central cylinders and end-cones.


Assuntos
Anabaena/metabolismo , Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas , Sequência de Aminoácidos , Anabaena/imunologia , Anabaena/ultraestrutura , Proteínas de Bactérias/imunologia , Imuno-Histoquímica , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia
11.
Mol Microbiol ; 2(5): 545-52, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3141741

RESUMO

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.


Assuntos
Proteínas Arqueais , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Membrana , Microcystis/análise , Organelas/análise , Proteínas , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Genes , Microcystis/citologia , Microcystis/genética , Dados de Sequência Molecular , Peso Molecular , Fenilalanina/análise , Prolina/análise , Mapeamento por Restrição , Tripsina/farmacologia
12.
Microbiology (Reading) ; 145 ( Pt 10): 2757-68, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10537197

RESUMO

Part of the gas vesicle gene cluster was amplified by PCR from three strains of Planktothrix rubescens isolated from Lake Zürich, Switzerland. Each contains multiple alternating copies of gvpA and gvpC. All of the gvpA sequences in the different strains are identical. There are two types of gvpC: gvpC20, of length 516 bp, encodes a 20 kDa protein of 172 amino acid residues (whose N-terminal amino acid sequence is homologous with the sequence of GvpC in Planktothrix [Oscillatoria] agardhii); gvpC16, of length 417 bp, encodes a 16 kDa protein of 139 amino acid residues that differs in lacking an internal 33-residue section. An untranslated 72 bp fragment from the 3' end of gvpC, designated omegaC, is also present in some strains. The two types of gvpC and presence of omegaC could be distinguished by the different lengths of PCR amplification products obtained using pairs of oligonucleotide primers homologous to internal sequences in gvpC and gvpA. Three genotype classes were found: GV1, containing only gvpC20; GV2, containing gvpC20 and omegaC; and GV3, containing gvpC16, gvpC20 and omegaC. Subclasses of GV2 and GV3 contained either one or two copies of omegaC. The accompanying paper by D. I. Bright & A. E. Walsby (Microbiology 145, 2769-2775) shows that strains of the GV3 genotype produce gas vesicles with a higher critical pressure than those of GV1 and GV2. A PCR survey of 185 clonal cultures of P. rubescens isolated from Lake Zürich revealed that 3 isolates were of genotype GV1, 73 were of GV2 and 109 were of GV3. The PCR technique was used to distinguish the gas vesicle genotype, and thence the associated critical-pressure phenotype, of single filaments selected from lakewater samples. Sequence analysis of the 16S rDNA and of regions within the operons encoding phycoerythrin, phycocyanin and Rubisco confirmed that these strains of Planktothrix form a tight phylogenetic group.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Cianobactérias/ultraestrutura , Genes Bacterianos , Variação Genética/genética , Proteínas de Membrana/genética , Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cianobactérias/isolamento & purificação , DNA Bacteriano/genética , Água Doce/microbiologia , Gases , Genótipo , Proteínas de Membrana/química , Dados de Sequência Molecular , Família Multigênica , Organelas/genética , Análise de Sequência de DNA , Suíça
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