Identification of arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein.

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants. and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.

[Obtaining transgenic Solanum tuberosum potato plants with active bacterial genes xyl and T-cyt effecting the phytohormone balance]. / Poluchenie transgennykh rastenii kartofelia Solanum tuberosum, nesushchikh aktivnye bakterial'nye geny xyl i T-cyt, vliiaiushchie na balans fitogormonov.

The genetic constructions based on integrative vector pGV3850 were used to introduce bacterial genes xyl and T-cyt into potato cells. The transformation was carried out using the leaf-disc method with modifications. A special system for obtaining regenerants from explants of potato in vitro plants or calli has been designed that permitted the selection of transgenic shoots. The presence of the genes in potato genome has been proved by testing the NPTII and glucoisomerase activities. The transgenic plants expressing T-cyt gene differed from the wild type in sharp decrease of the apical dominance.

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress.

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants. Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.

[Cloning of chloroplast psbA and rbcL-genes from cotton Gossipium hirsutum]. / Klonirovanie khloroplastnykh psbA i rbcL-genov khlopchatnika Gossipium hirsutum.

The fragments of cotton Gossipium hirsutum c.v. 108-f chloroplast genome were cloned in Escherichia coli cells. The cloned psbA and rbcL genes have been selected using the heterologous probes from spinach. The preliminary attempts to clone the complete psbA gene in pUC19 vector failed, probably, due to the toxicity of its product to Escherichia coli cells, and its 5'- and 3'-ends were cloned separately. Reconstruction experiments revealed that while the complete psbA gene was unable to be stably inherited by Escherichia coli cells, its structural part lacking the promoter region could be readily cloned in the bacterial cells.

[Transfer of the agrobacterial gene for cytokinin biosynthesis into tobacco plants]. / Perenos v rasteniia tabaka agrobakterial'nogo gena biosinteza tsitokinina.

The gene transfer into plants using the genetic engineering methods gives us the possibility to obtain transgeneric plants having acquired the new traits. Some bacterial genes can be used for this purpose. Obtaining of a transgeneric plant harbouring the cytokinin synthesis gene ipt (gene 4) from the T-DNA of Agrobacterium tumefaciens Ti-plasmid seems to be useful. The expression of tumor agrobacterial ipt gene in transformed plant cells interferes with the normal growth and regulation of the whole plant. The successful transfer of the cloned ipt gene from the recombinant plasmid pGV0319 into the tobacco plant using Agrobacterium vectors and succeeding regeneration of phenotypically normal transgenic plants are reported in the present paper.

[The study of binding of the virG gene product with the virD gene promotor region of the Agrobacterium tumefaciens Ti-plasmid C58]. / Izuchenie sviazyvaniia produkta gena virG s promotornoi oblast'iu gena virD Ti-plazmidy Agrobacterium tumefaciens C58.

The 1.5 kb EcoRI--HindIII fragment of the pTiC58 containing the virD regulatory sequence demonstrates a constitutive promoter activity in E. coli background and an inducible one in agrobacterium. The virG gene was cloned in pTZ19R plasmid. To reveal the virG product--virD regulatory sequence interaction a few protein fractions of E. coli harbouring the obtained recombinant plasmid pTZ19G lysate were used. PAGE-retardation assay revealed the specific binding between the 1.5 kb DNA fragment containing 5'-end of virD and a separate protein fraction of the bacterial lysate.

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski).

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.

[Electron microscopic and electron radioautographic study of cells in glandular hyperplasia and cancer of the endometrium]. / Elektronno-mikroskopicheskoe i élektronno- radioavtograficheskoe issledovanie kletok pri zhelezistoi giperplazii i rake éndometriia.

Peculiarities of the ultrastructure and RNA synthesis in the epithelial endometrial cells of nine patients with hyperplasia and the uterine cavity cancer have been studied by the electron-radioautographic method. Quantitative differences are revealed in the ultrastructures of the cells. Amplification of the nucleoli labelling and asynchronism of the RNA synthesis in the nucleoli with glandular hyperplasia and endometrial cancer are observed.

[Conjugation transfer of the bireplicon plasmid pSPA044 into Rhizobiaceae bacteria]. / Kon''iugatsionnyi perenos bireplikonnoi plazmidy pSPA044 v kletki bakterii semeistva Rhizobiaceae.

We have demonstrated the possibility of hybrid plasmid pSPA044 conjugative transfer from E. coli cells into different Rhizobium species. The bireplicon plasmid, constructed earlier in our laboratory, consisting of pBR325 and HindIII fragment 13 of the nopaline plasmid pTiC58 was mobilized for transfer by the helper plasmid pRK2013 with the frequency about 10(-4). We conclude the hybrid plasmid pSPA044 to be able to replicate stably in Rhizobiaceae cells.

[Integration of the mini-Mu phage into multicopy plasmids]. / Integratsiia mini-Mu-faga v mul'tikopiinye plazmidy.

Preparation of mini-Mu4 phage introduced into the pRP1.2 plasmid and carrying a 18 kb deletion of the central EcoRI fragment is described. The ability of the mini-Mu4 for independent transposition is demonstrated in experiments conducted for cointegrate formation (replicon fusion). The system developed is applicable to studying the mechanism of transposition, since the frequency of cointegrate formation may be descriptive of the transposition process. Frequency changes due to some factors may point to a possible influence of these factors on the mini-Mu transcription. The described mini-Mu integration into small multicopy plasmids of Col type makes it possible to conduct a simple physical analysis of structures formed in the course of transposition.

[Ultrastructural changes in hepatocyte nuclei reflecting genome expression and nuclear RNA biosynthesis in the early premitotic period of rat liver regeneration]. / Ul'trastrukturnye izmeneniia iader gepatotsitov, otrazhaiushchie ékspressiiu genoma, i biosintez iadernoi RNK v rannem predmitoticheskom periode regeneratsii pecheni krys.

Modifications of the nucleus ultrastructure and specific radioactivity of nuclear and cytoplasmic RNA from hepatocytes were studied at early stages of liver regeneration using 14C-orotic acid as a labelled precursor. It is shown that the first three hours after partial hepatectomy are characterized by mobilization of inherent abilities of the nucleus and initiation of chromatin activation. Then three hours after operation the activity of nucleolar and extranucleolar apparatus increases. By 12 hours chromatin associates with a nuclear membrane for preparing DNA replication.

[Chloroplast DNA cloning in Escherichia coli. II. The properties of the recombinant plasmids bearing the EcoRI fragments of pea chloroplast DNA and the cloning of the DNA sequences with rRNA genes]. / Klonirovanie DNK khloroplastov v Escherichia coli. Soobshchenie II. Nekotorye svoistva rekombinantnykh plazmid, nesushchikh EcoRI-fragmenty DNK khloroplastov gorokha, i klonirovanie posledovatel'nostei DNK s rRNK-genami.

Previously a method of selection of colicine-defective recombinant plasmids by mitomycin C was described. A series of recombinant plasmids (CPS) with various EcoRI-fragments of pea chloroplast DNA has been obtained. This paper describes some properties of cloned fragments replicated in Escherichia coli. The alkali stability of recombinant plasmid DNAs has been demonstrated, indicating the absence of ribonucleotides in their structure. Heterogeneity of chloroplast DNA in nucleotide composition was demonstrated using ultracentrifugation analysis of CPS-plasmid DNAs in CsCl-actinomycin D density gradient. Pea chloroplast rDNA was cloned in recombinant plasmids.

[Changes in hepatocyte ultrastructure under the influence of chronic furacillin administration to rats]. / Izmenennia ul'trastruktury gepatotsitov pod vliianiem khronicheskogo vvedeniia krysam furatsilina.

The ultrastructure of rat liver cells was studied during a 6 months' furacilline (5-nitro-2-furtfurilsemicarbazon) feeding in dose of 40 mg per day, and also during the next 8 months after the treatment cessation. An irregular swelling of membranous structure in addition to disorganization and partial reduction of the granular endoplasmic reticulum were found in the hepatocyte cytoplasm after the prolonged furacilline feeding as well as glycogen depletion and the tendency of the agranular endoplasmic reticulum to enlargement. These changes rarely reached the essential intensity with the transition to necrobiosis. They disappeared already a month after the cessation of furacilline treatment. In all the terms of experiment, the nucleoli were hypertrophied and retained their loose nucleoloneme structure. No sings of furacilline carcinogenic activity were found in rat liver during the 14 months of investigation.

[Cloning chloroplast DNA in escherichia coli. I. Construction and selection of recombinant plasmids containing fragments of pea chloroplast DNA]. / Klonirovanie DNK khloroplastov v Escherichia coli. Soobshchenie I. Konstruktsiia i selektsiia rekombinantnykh plazmid, soderzhashchikh fragmenty DNK khloroplastov gorokha.

Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained.