Biomedical subjects
Publications and source records attributed to M I Mendzhul.
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.
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.
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Three methods of plasmids isolation from the cells of cyanobacteria: Plectonema boryanum 465, P. nostocorum 636, P. nostocorum 638, P. edaphicum 262, Plectonema sp. 456, Phormidium uncinatum 528, Anabaena variabilis 458 and Synechococcus cedrorum 750 have been approved. The method of plasmids isolation by 1 M NaCl salting out was used for plasmid screening. All the strains studied contained large, above 55 KB plasmids, small plasmids (1.5 KB) were found in the cells of P. boryanum 465 and P. nostocorum 636. After the restriction analysis a small plasmid of P. boryanum 465 was chosen to be used in the experiments on the vector construction. The above plasmid was called pSM1 was cloned into the vector pBluescri ptIISK(+) on the unique ClaI site. The obtained construction pBSMI may be used as the basic one for creation of cyanobacterial vectors.
The activity level and some physico-chemical properties of enzymes of the tricarboxylic acid cycle (TCA cycle) and the associated enzymes isocitrate lyase and glutamate dehydrogenase of cyanobacterium Spirulina platensis grown under illumination of 5000 lk in batch conditions, have been studied. High activities of most of the studied enzymes except for alpha-ketoglutarate dehydrogenase (alpha-KGDH) and succinate dehydrogenase have been estimated. In some cases the activities were by an order higher than that of similar enzymes in other cyanobacteria. This reflects the microorganism ability to synthesize intensively organic substances and first of all protein. Absence of alpha-KGDH activity proves that TCA cycle of spirulina has a limited value for energy generation and mainly performs the biosynthetic function.
A physical map of plasmid pSM1 has been compiled as a result of restriction analysis of pBSMI isolated from the cells of cyanobacterium Plectonema boryanum 465. A comparative analysis of the restriction sites localization in DNA of plasmid pSM1 with site topology of plasmids pGL3 of P. boryanum and pPF1 of Phormidium foveolarum indicates to high degree of these plasmids homology.
The associative cultures of cyanobacteria were selected which may be used for development of technology of tertiary treatment of sewage for yeast and alcohol production plants. Growth parameters of associations were studied during periodic and continuous cultivation. When studying the influence of flowing speed for some exponents of treatment it was shown that cultivation of cyanobacteria should be carried out with the change of volume not more than 0.01 h. Final treatment 0.010 h-1 in order to return sewage to natural water reservoirs may be carried out by means of Oscillatoria sp. in bioponds.
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.
A possibility to use terminal transferase to obtain hybridized RNA-probes has been investigated. Optimal conditions of the reaction proceeding were elaborated on the model objects--RNA of the phage MS2: 200 mM of potassium cacodylate, pH 7.2, 1 mM CoCl2, 1 mM DTT, under the considerable surplus of the enzyme. The elaborated method was used to determine the area of early genes of cyanophage LPP-3. With this purpose total RNA preparations were isolated from cyanobacterium Plectonema boryanum CALU 465. The above preparations were labeled by means of terminal transferase and [alpha-32P] ATP and used as the probes in hybridization with the DNA restriction fragments of LPP-3. Proceeding from the results of Southern-blots, a conclusion was made, that the KpnI-fragment of genome of cyanophage LPP3 10 kb in size includes the early genes. A possibility of practical use of the developed method is discussed.
The efficiency of radioactive labeling of 3'- and 5'-ends of cyanophage LPP-3 DNA by polynucleotide kinase T4 and terminal transferase under various reaction conditions has been investigated. The obtained data prove that cyanophage LPP-3 DNA has the protruding 3'-ends. The experiments on ligation of native molecules of LPP-3 DNA evidence that the virus genome ends do not display any complimentarity. Separate fragments of LPP-3 DNA were cloned. The restriction analysis of the cloned fragments has confirmed a supposition on the absence of LPP-3 cyanophage of GGGCC and GGCCC sequences in the genome. A hypothesis has been suggested about similar site-specificity of the virus. Counterselection of the genome LPP-3 cyanophage allows it to be considered a promising one in the construction of new cloning vectors in cyanobacterium.
Restrictases fit for the purposes of physical mapping of cyanophage LPP-3 DNA have been selected as a result of the restriction analysis. The use of the methods of mutual hydrolysis, restriction of the fragment isolated from gel and terminal labeling allowed formation a physical map of LPP-3 cyanophage DNA with the complete scheme of allocation of 14 sites for 8 restrictases: Alw44I, Bsp1191, BsuRI, Eco147I, EheI, NcoI, Kpn2I and PvuI as well as the position of certain sites for restrictases HindIII, KpnI and Sau3A.
Screening of cyanobacteria cultures for their sensitivity to the action of complex preparations of toxins from 18 strains of Stachybotrys chartarum has been carried out with the purpose to search for new test-organisms to create a reliable system of biotesting of stachibotryotoxines. In contrast to other cultures of unicell (Anacystis nidulans) and filamentous (Anabaena sp., Anabaena variabilis, Nostoc linckia, Plectonema boryanum) cyanobacteria used in the experiment, only one culture of unicell cyanobacterium Synechococcus cedrorum proved to be highly sensitive to the action of preparations of the whole group of studied mycotoxins. Their activity with respect to this culture varied within wide limits (lysis zone diameter is 11-30 mm) and for the most of strains it coincided with or exceeded this index determined with the use of other biotests. Owing to the use of Synechococcus cedrorum as test culture the authors have proved for the first time the toxicity of three strains of Stachybotrys chartarum (K15822, 14722, 14186). Basing on the data obtained the authors have determined the culture of the unicell cyanobacterium, according to the sign of high sensitivity to all the studied stachibotryotoxin preparations of solid and liquid nutrient media, as a new test-organism for detection of this group of toxins and it may be recommended for practical use. Optimal conditions for growing test-culture and biotesting of toxins have been defined.
The paper deals with the basic trends of fundamental investigations of the Department of Algae Viruses in the field of cyanophagia-ecology, biological and physico-chemical properties of cyanophages as well as interrelation with the host cells. Such problems as a possibility to use the system cyanophage-cyanobacteria as the experimental model for development of the unified functional model of productive infection, efficient methods of prophylaxis and therapy of virus infections as well as the solution of various biotechnological problems are discussed.
Physico-chemical and catalytic properties of proteinases of native and induced cells of cyanobacteria Plectonema boryanum have been comparatively studied. It has been established that at early stages of reproduction of cyanophage LPP-3 in cyanobacteria P. boryanum is formed de novo proteinase complex consisting at least of five enzymes. Proteinases induced by the virus are distinguished from those of native cells by a series of physico-chemical characteristics and possess higher catalytic activity. Analogous virus-induced changes in proteinase complex also occur in the system cyanobacterium Anabaena variabilis--cyanophage A-1. Possible functions of certain enzymes of proteinase complex in the virus pathology of cyanobacteria cells are discussed in the paper.