RESUMO
Three vector constructions--pSTS2, pSTS3 and pSTS4 have been obtained on the basis of the standard vector for Escherichia coli pBR 322, cyanobacterial plasmid pSM1 and its separate fragments. The structure of pSTS4, carrying the full-size plasmid pSM1 (cloned by the restriction site Hpa II), replicated both in the cells of E. coli and in the cells-transconjugants of the trichomal cyanobacterium Plectonema boryanum. Under these conditions resistance of P. boryanum cells to ampicillin increases three orders, and to tetracyclin--50 times. Structural integrity of pSTS4 is stably supported in conjugates during seven successive passages (time of the experiment) and preserved the transforming capacity in respect of the cells of E. coli. It is supposed that pSTS4 is a shuttle-vector with two active origins of replication; one of them provides for the replication of pSTS4 in E. coli cells, another--in P. boryanum cells.
Assuntos
Cianobactérias/genética , Vetores Genéticos , Ampicilina/farmacologia , Antibacterianos/farmacologia , Clonagem Molecular , Conjugação Genética , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Técnicas de Transferência de Genes , Plasmídeos , Mapeamento por Restrição , Tetraciclina/farmacologia , Transformação BacterianaRESUMO
The nucleotide sequence was established for the rep gene of plasmid pSM1 isolated from cyanobacterium Plectonema boryanum CALU 465. Both nucleotide sequence and the encoded amino acid sequences showed 98% homology to the corresponding sequences of small plasmids pPF1, pGL3, pPBS1, pBLX, and pPB1. An active center was identified in the replicative protein sequences.
Assuntos
Cianobactérias/genética , Genes Bacterianos , Plasmídeos , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.
Assuntos
Celulose/análogos & derivados , Cianobactérias/enzimologia , Desoxirribonucleases/isolamento & purificação , Centrifugação , Desoxirribonucleases/química , Etanolaminas , Troca Iônica , Peso Molecular , SacaroseRESUMO
The plasmid composition of the Plectonema boryanum Gom. Cyanobacterium, strain CALU 465, was analyzed. A small pSM1 plasmid, size 1.5, was chosen for genetic engineering. The physical map, constructed for the plasmid, was used to create the pSTS series, i.e. vector construction for the P. boryanum cyanobacterium. A method was selected and tested for the introduction of the vector into host cells. The needed orientation of pSM1 cyanobacterium plasmid within the vector as well as a possible role of its separate elements in inheriting the construction by host were defined.
Assuntos
Cianobactérias/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Plasmídeos/genética , Ampicilina/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Conjugação Genética , Cianobactérias/efeitos dos fármacos , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Ordem dos Genes , Canamicina/farmacologia , Plasmídeos/metabolismo , Mapeamento por Restrição , Estreptomicina/farmacologia , Tetraciclina/farmacologiaRESUMO
It was shown in experiments carried out on cyanobacterium P. boryanum, that when cells are incubated in a buffer with low concentrations of nonion detergent (0.1%, Triton X-100) one can observe the DNAse activity release into the medium. The methods of supersound desintegration of cells and following gradient centrifugation of the cell-free extracts were used to obtain three fractions of the cell membranes identified by means of spectrophotometric analysis. It has been established that P. boryanum is characterised by high level of DNAse activity associated with the cytoplasmic cell membrane. As to its activity character the DNAse under study belongs to the class of nonspecific endonucleases.
Assuntos
Cianobactérias/metabolismo , Desoxirribonucleases/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Cianobactérias/enzimologia , Citoplasma/enzimologia , Desoxirribonucleases/análise , Espectrofotometria , Frações Subcelulares/enzimologiaRESUMO
It has been shown that cells of trichoma cyanobacterium Plectonema boryanum G o m. strain CALU 465 do not possess natural resistance to antibiotics ampicyllin, chloramphenicol, streptomycin and canamycin. A shuttle vector pSTS1 has been constructed on the basis of cyanobacterial plasmid pSM1 and vector pBR 322. Conditions have been developed and conjugative transfer of pSTS1 to the cells of cyanobacterium P. boryanum has been performed. As the result of expression of beta-lactamase gene pSTS1 the resistance of cells-conjugantes to ampicillin increased by three orders.
