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[Spatial organization of replicons in the eukaryotic nucleus: attachment of replication initiation regions to the nuclear skeleton].

The DNA fraction enriched in replication origins was isolated from chicken erythroblastosis cells with the aid of nascent DNA strand extrusion method. In renaturation experiments, the sequence specificity of this DNA fraction (oriDNA) was compared to the sequence specificity of the nuclear matrix DNA. The oriDNA was shown to comprise a specific subset of unique genes all of which being represented also in the nuclear matrix DNA. The same unique DNA sequences were found to be depleted from the oriDNA and from the nuclear matrix DNA. A conclusion has been drawn that replication origins are located at the basements of DNA loops. The putative positions of replication origins within the domain of chicken alpha globin genes were mapped by hybridization experiments and were found to coincide with the positions of stable (not depending on the transcriptional state of globin genes) sites of DNA attachment to the nuclear skeleton.

Animals↗

[Use of the plasmid R89S replicon for constructing integration vectors].

It has been shown that the plasmid R89S derivatives can be used as integrative vectors for bacteria in which the plasmid is unable to replicate autonomously. The chromosomal and plasmid fragments of phototrophic bacterium Rhodobacter sphaeroides have been cloned in plasmid pVZ365, a SmRKmR-derivative of R89S. The obtained recombinant plasmids were mobilized into R. sphaeroides cells by the I pcP-group conjugative plasmid R751. The frequencies of the SmR-transconjugants formation are 3.7.10(-5) to 5.6.10(-3) per recipient cell. The formation of the SmR-transconjugants has not been revealed in case of the plasmid pVZ365 mobilization. The recombinant molecules containing R. sphaeroides plasmid fragments have been shown to integrate into endogenous plasmids and form cointegrates with them.

Genetic Vectors↗

[A new vector for cloning of genes and replicons in yeasts].

A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.

Cloning, Molecular↗

[Caffeine inactivation of mammalian cells in the S phase of the mitotic cycle due to anomalous replicon initiation during the inhibition of the elongation of replicating DNA].

Chinese hamster cells were treated with an inhibitor of DNA synthesis (hydroxyurea or arabinoside-cytesine) in non-toxic concentrations for 20 hours in the presence or absence of caffeine (2 mM). Under these conditions caffeine considerably inactivates the cells. If cells are synchronized by hydroxyurea (0.25 mM) in the S-phase of mitotic cycle, the addition of caffeine kills all the S-phase cells, while gamma-irradiation or novobiocine treatment markedly decreases the sensibilizing effect of caffeine. These findings permit us to conclude that cell inactivation is due to anomalous reinitiation of DNA synthesis stimulated by caffeine in the presence of drugs which inhibit the DNA chain elongation.

Animals↗

New approaches for mucosal vaccines for AIDS: encapsidation and serial passages of poliovirus replicons that express HIV-1 proteins on infection.

It is apparent that a safe and effective HIV vaccine is an important component in the development of rational approaches for the control and prevention of HIV transmission. Given the fact that the virus most often encounters a mucosal surface during sexual transmission, a vaccine designed to stimulate both the systemic and mucosal immune systems is essential. Poliovirus is attractive as a delivery system because of several biological features inherent to the virus. First, the pathogenesis of the virus has been well studied, and important features have been identified. The virus is naturally transmitted by a fecal-oral route and is stable in the harsh conditions of the gastrointestinal tract. Second, previous studies using attenuated vaccine strains of poliovirus showed that a long-lasting systemic and mucosal immunity is generated after administration of the vaccines. Studies have demonstrated the presence of circulating T cells that proliferate to whole inactive poliovirus or peptides corresponding to amino acids of the VP1 proteins in previously immunized individuals. These results established that immunization with poliovirus stimulates both the humoral and cell-mediated components of the immune system. Third, the attenuated strains of poliovirus are safe for humans and are given to infants as early as 6 months of age. The incorporation of foreign genes into the attenuated strains would be an attractive feature that should pose no more of a health risk than that associated with administration of the attenuated vaccines. Finally, studies from this laboratory, as well as others, have established the feasibility of incorporating foreign genes into the poliovirus cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Vaccines↗

Transfer of hybrid plasmids based on the replicon pRRI7 from Escherichia coli to Bacteroides and Prevotella strains.

New shuttle vectors based on a Prevotella ruminicola 9.5 kb cryptic plasmid (pRRI7) inserted within the Escherichia coli vector pKC71, carrying the Ccr/Emr Bacteroides marker, were constructed. These constructs (pKBR23-1 and pKBR23-2) were transferred into Bacteriodes distasonis, Bacteroides thetaiotaomicron, Bacteroides uniformis and into P. ruminicola NCFB 2202 either by conjugal mobilization or by electroporation. Another pRRI7 derivative based on pKC72, pKBR23-3, was smaller (13.1 kb) and non-mobilizable. By electroporation, it was transferred to Bact. distasonis and P. ruminicola. Being derived from pRRI7 which is compatible with the shuttle plasmid pRRI207, the host/vector combination involving P. ruminicola NCFB 2202 and pKBR23-3 offers new possibilities for genetic investigations in rumen anaerobic bacteria after further introduction of a second readily selectable marker within pRRI207 or pKBR23-3.

Bacteroides↗

A size analysis of the adenovirus replicon.

The linear double-stranded genome of adenovirus DNA replicates semiconservatively from two origins of replication at either of the two molecular ends. Using an in vitro replication system which is able to initiate de novo DNA synthesis we have mapped the origin of DNA replication within the terminal 19 bp of the viral genome. Our conclusions are based on the use of different natural DNA templates, i.e., adenovirus type 2 and mouse adenovirus Fl DNA. In addition, we have employed linearized plasmid DNA templates which contain cloned terminal restriction enzyme fragments as well as chemically synthesized adenovirus termini of different length.

Adenoviridae↗

The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

Basidiomycota↗