RESUMEN
The cytotoxic effect of culture filtrates from healthy, heat-stressed, and repaired Listeria monocytogenes and L. innocua on the bovine mammary epithelial cell line MAC-T was examined. Culture filtrates were collected from Listeria spp. following treatments which included: (i) 18 h of growth of Listeria at 37°C; (ii) sublethal heat treatment at 56°C for 50 minutes; (iii) repair of the injured cells at 37°C for 7 h; (iv) growth of repaired bacterial cells at 37°C for 36 h; and (v) heat injury at 56°C for 50 min of the cell population obtained after the initial repair and growth. Strains chosen for study included two genetic mutants of L. monocytogenes : a hemolysin-negative mutant, CNL 85/162 (Hly-) and a hemolysin-positive revertant, CNL 85/163 (Hly+). Culture filtrates obtained from Hly- bacteria did not prevent adhesion of the mammary epithelial cells and slightly stimulated their growth. In contrast, culture filtrates from Hly+ bacteria grown for 18 h significantly reduced the ability of MAC-T cells to adhere to the cell culture dishes, prevented the growth of those cells that were attached to the dishes, and caused cell death. Supernatants from Hly+ and Hly- following injury and during repair had no lethal effect on MAC-T cells. The effects of culture medium obtained after growth of the repaired Listeria cells on MAC-T cells were similar to those recorded for medium from the first 18 h growth for both strains, indicating that cells regain virulence potential once they have repaired and reinitiated growth. Culture filtrates obtained from L. monocytogenes Scott A showed results similar to those of Hly+, decreasing adherence and growth of MAC-T cells, while L. innocua culture filtrate had no adverse effect. The results of these experiments suggest that when injured, L. monocytogenes does not demonstrate adverse effects towards MAC-T cells. Once repair is completed and the listeria are growing, activity towards MAC-T cells is restored.
RESUMEN
Human corneal endothelial cells (HCEC) were transfected with some cloned oncogenes. The direct microinjection of either early region (E1) genes of monkey (SA7) and human (Ad5) adenoviruses or Ha-ras oncogen in conjunction with the Ad5 Ela-gene into embryonic HCEC nuclei was shown to result in immortalization of these cells. 3 independent immortalized HCEC lines were established in their growth and morphological properties were studied. These properties were very similar to those of primary HCEC, but unlike primary HCEC the immortalized cells didn't need the endothelial cell growth factor.
Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Endotelio Corneal/citología , Oncogenes , Adenovirus Humanos/genética , Adenovirus de los Simios/genética , Animales , Línea Celular Transformada , Humanos , Técnicas In Vitro , Microinyecciones , TransfecciónRESUMEN
The technique is described for obtaining pH-sensitive biotinylated liposomes. The liposomes included phosphatidyl ethanolamine, oleic acid, cholesterol, biotin amidocaproyl phosphatidyl ethanolamine, with a molar ratio of 7:3:3:0.1 or 4:2:4:0.1. The fluorescein stain calcein at ++self-limited concentration, 32-P-dTTP, or DNA was incorporated into the internal liposome medium. The liposomes were stable in neutral or mildly alkaline medium but became leaky and showed agglutination at pH 5.7. Liposomes were agglutinated in the presence of avidin and bound specifically to the layer of avidin on the surface of immunological plates. The apparent liposome affinity to the avidin layer was up to 10(-11) M. The avidin-biotin system allows for specific binding of liposomes to human lymphocytes via biotinylated anti-CD5-antibodies. A simple, convenient, and rapid procedure has been elaborated for the binding of water-soluble substances (plasmid DNA included) to liposomes without DNA destruction.
Asunto(s)
Avidina/farmacología , Biotecnología/métodos , Biotina/farmacología , ADN/administración & dosificación , Liposomas/administración & dosificación , Linfocitos/efectos de los fármacos , Fosfatidiletanolaminas/farmacología , Plásmidos , Sitios de Unión , ADN/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Linfocitos/metabolismoRESUMEN
Microinjection of either type 1 human adenovirus, type SA7 monkey adenovirus virions or circular adenovirus DNA, obtained by the treatment of DNA-terminal protein complexes with glutaraldehyde, into nuclei of permissive cells results in the complete cycle of virus reproduction. Microinjection of neither linear native, condensed adenovirus DNA nor the DNA-terminal protein complexes under the same conditions initiates the adenovirus reproduction thought the synthesis of early and some late viral antigens is observed in the injected cells. Integration of injected adenovirus DNA into the cellular DNA occurs as far as 30 min after injection. Microinjection of either adenovirus DNA or its oncogene containing fragments into nuclei of semipermissive cells induces the transformation of these cells. In this case the time of the first appearance of transformation foci is decreased.
Asunto(s)
Adenoviridae/genética , ADN Recombinante/genética , ADN Viral/genética , Transfección , Virión/genética , Animales , Autorradiografía , Transformación Celular Neoplásica/genética , Transformación Celular Viral/genética , Células Cultivadas , ADN Viral/ultraestructura , Electroforesis en Gel de Agar , Técnica del Anticuerpo Fluorescente , Humanos , Microinyecciones , Hibridación de Ácido Nucleico , Plásmidos , Timidina Quinasa/genética , Proteínas Virales/metabolismoRESUMEN
EV40 DNA closed superhelical form was introduced into cell nuclei by means of direct microinjection and expression of the viral genome followed. The expression of viral T-antigen was observed 40 hours after the injection, and SV40-specific effect developed on the fifth day. Using the method discussed, the infection dose of SV40 DNA in a permissive cell culture system was shown to be approximately one molecule per cell nucleus.
Asunto(s)
Transformación Celular Viral , ADN Viral/genética , Genes Virales , Virus 40 de los Simios/genética , Animales , Antígenos Virales de Tumores/genética , Línea Celular , Núcleo Celular/metabolismo , ADN Superhelicoidal/genética , Haplorrinos , Cinética , Microinyecciones , Virus 40 de los Simios/inmunologíaAsunto(s)
Adenoviridae/genética , Adenovirus Humanos/genética , Adenovirus de los Simios/genética , Genes Virales , Adenovirus Humanos/ultraestructura , Adenovirus de los Simios/ultraestructura , ADN Viral/análisis , ADN Viral/genética , Microscopía Electrónica , Ácidos Nucleicos Heterodúplex/análisis , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
The effect at specific nuclease S1 on DNA and the complex viral DNA-terminal protein of the highly oncogenic simian adenovirus SA7(C8) was studied. It was shown that nuclease S1 did not digest the bound between DNA and terminal protein in the complex but residual amino acid(s) was cleaved out after digestion with pronase. The DNA obtained after nuclease S1 action could be ligated and its 5'-ends were phosphorylated by polynucleotide kinase.
Asunto(s)
Adenoviridae/análisis , Adenovirus de los Simios/análisis , ADN Viral/análisis , Endonucleasas , Enzimas de Restricción del ADN , Desoxirribonucleoproteínas/análisis , Pronasa , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
The physical map of human adenovirus type 1 DNA was constructed with the Hind III restriction enzyme. Direct comparison of the DNA fragments with those of adenovirus type 2 revealed that the genome of adenovirus type 1 is 200 to 300 base pairs longer. The difference are located outside the region of the inverted terminal repetition within three distinct loci. Fragment Hind III-F of type 1 DNA which is presumed to carry all the genes responsible for in vitro transformation can be separated without considerable contamination by a single electrophoretic step.
Asunto(s)
Adenovirus Humanos/genética , ADN Viral/análisis , Composición de Base , Inversión Cromosómica , Mapeo Cromosómico , Replicación del ADN , Enzimas de Restricción del ADN/metabolismo , ADN Viral/genéticaAsunto(s)
Adenoviridae/genética , Adenovirus de los Simios/genética , Proteínas de la Cápside , Cápside/análisis , ADN Viral/análisis , Genes Virales , Péptidos/análisis , Proteínas Virales/análisis , Animales , Línea Celular , Chlorocebus aethiops , Mapeo Cromosómico , Humanos , Ácidos Nucleicos Heterodúplex , Hibridación de Ácido NucleicoRESUMEN
The effect of restricting R. BgIII and R. HindIII endonucleases on the genome of simian adenovirus type 7 (SA7) was studied. The R. BgIII endonuclease has 5 restriction sites on SA7 DNA and produces 6 fragments with molecular weights of 10.0 X 10(6), 5,3 X 10(6), 3.6 X 10(6), 1.8 X 10(6), 0.6 X 10(6), and 0.5 X 10(6) daltons. R. HindIII, having 3 restriction sites, hydrolyses SA7 DNA into 4 fragments with molecular weights of 7.6 X 10(6), 5.9 X 10(6), 5,2 X 10(6), 3,1 X 10(6) daltons. The R. BgIII and R. HindIII fragments are arranged in the SA7 genome in the following order: B-A-C-E-D-F and C-D-B-A.
Asunto(s)
Adenoviridae/ultraestructura , Adenovirus de los Simios/ultraestructura , Enzimas de Restricción del ADN/metabolismo , ADN Viral/metabolismo , Secuencia de Bases , ADN Viral/análisis , Genes Virales/efectos de los fármacos , Hidrólisis , Peso MolecularRESUMEN
The effect of 7 specific endonucleases EcoRI, BamHI, BglII, SalI HindIII, XhoI, SmaI, on the genome of the phage Sd was studied. The molecular weight of the genome was estimated as 45.10(6). BamHI, EcoRI, HindIII, BglII, SmaI have altogether 15 sites of restriction SalI and XhoI do not hydrolyse the phage DNA. Fragmentation of the phage DNA in conditions of partial hydrolysis and simultaneous action of several enzymes have allowed to draw a physical man to the phage Sd DNA is more resistent to the action of restriction enzymes than DNAs from other phages. The mean size of Sd DNA fragments exceeds the statistical value almost by one order of magnitude.
Asunto(s)
Colifagos/genética , Genes Virales , Mapeo Cromosómico , Enzimas de Restricción del ADN , ADN Viral , Hidrólisis , Peso MolecularRESUMEN
The restricting endonuclease R. EcoRI hydrolyses human adenovirus type I (Adl) DNA with formation of three fragments with molecular weights of 17.5 x 10(6), 3.7 x 10(6), and 1.55 x 10(6) dalton. Under the effect of R. SaLI restrictase DNA Adl disintegrates into 5 fragments with molecular weights: 8.0 x 10(6), 6.0 x 10(6), 4.6 x 10(6), 4.0 x 10(6), and 0.2 x 10(6) dalton. Treatment of DNA with R. BamHI produces 4 fragments with molecular weights 10.0 x 10(6), 6.8 x 10(6), 4.0 x 10(6), and 2.2 x 10(6) dalton. Fragments of R. SaLI and R. BamHI form sequences BECAD and ACBD, respectively.
Asunto(s)
Adenovirus Humanos/análisis , Enzimas de Restricción del ADN , ADN Viral , Catálisis , Fenómenos Químicos , Química , Genes Virales , HidrólisisRESUMEN
A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.
Asunto(s)
Enzimas de Restricción del ADN/aislamiento & purificación , ADN Recombinante , Cloromercuribenzoatos/farmacología , Colifagos/enzimología , Enzimas de Restricción del ADN/antagonistas & inhibidores , Enzimas de Restricción del ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Escherichia coli/enzimología , Glicerol/farmacología , Concentración de Iones de HidrógenoRESUMEN
The effect of the restricting endonucleases R.EcoRI, R.BamI and R.SalI on the genome of type 7 simian adenovirus (SA-7) has been studied. Since the DNA has only one site of R.EcoRI recognition the enzyme cleaves SA-7 DNA into two fragments with the molecular weights 12.0 and 10.0 . 10(6). The restrictase R.BamI cleaves the SA-7 DNA at six sites producing 7 fragments with the molecular weights 6.6, 5.9, 3.8, 2.7, 1.3, 0.7 and 0.6 . 10(6). R.SalI cleavage yields 6 fragments with the molecular weights 8.1, 5.5, 4.3, 2.45, 1.2 and 0.6 . 10(6). The R.BamI and R.SalI fragments are arranged in the orders E-A-D-F-C-G-B and A-B-D-F-E-C, respectively. The only R.EcoRI recognition site is localized in the C fragment produced by R.BamI and in the B fragment produced by R.SalI.
Asunto(s)
Adenoviridae/genética , Adenovirus de los Simios/genética , ADN Viral/genética , Mapeo Cromosómico , Enzimas de Restricción del ADN/metabolismo , Peso MolecularRESUMEN
The effect of restrictive endonucleases R. Ecor I, R. Bam I, and R. Sal I on simian adenovirus type 7 (SA7) genome was studied. Because of the occurrence of the only recognition site, the R. EcoR I enzyme cuts SA7 DNA into two fragments with molecular mass of 12 and 10 X 10(6) daltons. R. Ram I cuts this DNA in 6 sites producing 7 fragments with molecular mass 6.6, 5.9, 3.8, 2.7, 1.3, 0.7 and 0.6 X 10(6) daltons. Treatment with R. Sal I gives 6 fragments with molecular mass of 8.1, 5.5, 4.3, 2.45, 1.2, and 0.6 X 10(6) daltons R. Bam I and R. Sal I fragments form the sequence E--A--D--F--C--G--B and A--B--D--F--E--C, respectively. The only recognition site of R. EcoR I is localized in the C fragment of R. Bam I and B fragment of R. Sal I.
Asunto(s)
Adenoviridae , Adenovirus de los Simios , ADN Viral/análisis , Endonucleasas , Escherichia coli/enzimología , Adenoviridae/genética , Adenovirus de los Simios/genética , Animales , Fraccionamiento Químico , Cricetinae , Electroforesis en Gel de Agar , Endonucleasas/farmacología , Genes Virales/efectos de los fármacos , Haplorrinos , Hidrólisis , Peso MolecularRESUMEN
The restriction endonuclease EcoRI hydrolyzes DNA to a greater number of fragments in the presence of glycerol than under normal conditions. This enzyme begins to work by the so-called EcoRI-type of restriction when glycerol concentration reaches 50%. The EcoRI activity appeared in experiments only when the ionic strength of the solution was decreased and pH of the solution was increased. However, under such extreme conditions the enzyme was quickly inactivated and it was difficult to obtain reproducible results especially for hydrolysis of the high-molecular DNA. The suggested conditions for the EcoRI activity permit to obtain reproducible results, this being practically equivalent to discovery of the new restriction endonuclease.
Asunto(s)
Enzimas de Restricción del ADN , Endonucleasas , Escherichia coli/enzimología , Glicerol , Adenovirus Humanos/análisis , Catálisis , Colifagos , ADN Viral , PlásmidosRESUMEN
The effect of restricting endonucleases of E. co RI and BAMI on DNA of human adenovirus type 6 was studied. E. co RI and BAMI restrictases cleave virus DNA into 4 fragments each. The molecular weight of E. co RI fragments was: A--17.5X10(6), B--3.63X10(6), C--1.56X10(6), D--1.05X10(6), those of BAMI fragments: A--9.7X10(6), B--7X10(6), C--4.09X10(6), D--3.1X10(6). By means of terminal nucleotidyltransferase and the procedure of partial DNA hydrolysis, the alteration of fragments obtained by the effect of E. co RI enzyme in adenovirus type 6 genome was determined and found to be A--D--C--B.