RESUMEN

MCF-7, a breast cancer-derived cell line, is deficient of caspase 3 and relatively insensitive to many chemotherapeutic agents. To study the association of caspase 3 deficiency and chemotherapeutic resistance, we reconstituted caspase 3 in MCF-7 cells and characterized their apoptotic response to doxorubicin and etoposide. Western blots demonstrated that caspase 3 was constitutively expressed in the reconstituted MCF-7 cells. Both morphological observation and survival assays showed that caspase 3 reconstitution significantly sensitized MCF-7 cells to both drugs. Remarkably increased activation of caspases 3, 6, and 7, cleavage of cellular death substrates, and DNA fragmentation were detected in the reconstituted MCF-7 cells after drug treatment. Together, these data demonstrated a specific role for caspase 3 in chemotherapy-induced apoptosis and in activation of caspases 6 and 7. Our results also suggest that caspase 3 deficiency may contribute to chemotherapeutic resistance in breast cancer.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Caspasas/fisiología , Doxorrubicina/farmacología , Etopósido/farmacología , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Caspasa 3 , Caspasas/biosíntesis , Caspasas/genética , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática , Humanos , Concentración 50 Inhibidora , Células Tumorales Cultivadas

RESUMEN

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G(1)/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G(1) arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

Asunto(s)

Ciclo Celular/fisiología , Factor de Crecimiento Nervioso/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Secuencia de Bases , División Celular/fisiología , Cartilla de ADN , Células PC12 , Ratas , Fracciones Subcelulares/metabolismo

RESUMEN

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

Asunto(s)

Antígenos Transformadores de Poliomavirus/análisis , Ciclo Celular/fisiología , Proteína p53 Supresora de Tumor/análisis , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada/química , Línea Celular Transformada/citología , Línea Celular Transformada/virología , ADN/análisis , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica , Immunoblotting , Inmunofenotipificación , Ratones , Proteína p53 Supresora de Tumor/genética

RESUMEN

To examine the pattern of retroviral vector gene expression during early stages of infection, a Moloney murine leukemia virus (M-MuLV)-based vector that transcribes the simian virus 40 (SV40) large T antigen (Tag) gene from the viral long terminal repeat (LTR) was used to infect proliferating rodent fibroblasts. At various times after infection, cells were fixed and stained for Tag by indirect immunofluorescence, and for DNA using propidium iodide. Tag immunofluorescence and DNA content were both quantified by flow cytometry. The results showed that Tag expression was first detected exclusively in the late G1 and early S phases of the cell cycle, approximately 12 h postinfection. The infection was synchronous in that Tag-expressing cells detected at 12 h, in late G1 and early S, moved as a discrete population through S phase, into the G2 + M phases of the cell cycle and then back into G1 during the next 8-10 h. The presence of a synchronous Tag-expressing cell population suggests that, at time of infection, cells in certain phases of the cell cycle were more susceptible to infection than cells in other phases. This may be related to synchronizing events that must occur before viral genes are expressed in infected cells; one such event may be integration of viral DNA into cellular chromosomes (i.e., provirus formation) that requires cells to pass through an M phase.

Asunto(s)

Ciclo Celular , Expresión Génica , Vectores Genéticos , Virus de la Leucemia Murina de Moloney/genética , Células 3T3 , Animales , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , ADN/metabolismo , Citometría de Flujo , Genes Virales , Ratones , Modelos Biológicos , Virus de la Leucemia Murina de Moloney/inmunología , Virus de la Leucemia Murina de Moloney/patogenicidad

RESUMEN

E2F transcription factors regulate expression of a panel of cellular genes that control cellular DNA synthesis and proliferation, either by activating or repressing their transcription, largely in a cell cycle-dependent manner. The ability of E2F proteins to regulate expression of these target genes is, in turn, regulated by other cellular proteins that are important for normal control of cell cycle progression. Together, E2F proteins, their target genes, and the proteins that regulate E2F activity comprise a genetic pathway that is probably the most, frequently altered pathway in human cancer. This review examines this genetic pathway and focuses on the role of E2F proteins in its function. Specifically, the target genes regulated by E2F, the likely mechanisms by which activation and repression of target gene transcription is achieved, and the regulation of E2F activity by other proteins in the cell, are discussed.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/metabolismo , División Celular/genética , Factores de Transcripción E2F , Humanos , Neoplasias/etiología , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Transcripción Genética

RESUMEN

In cells overexpressing an exogenous cDNA encoding the E2F-1 transcription factor, as many as eight closely-migrating protein bands were detected after denaturing protein gel electrophoresis and immunoblotting with an E2F-1-specific antibody. Control cells, not overexpressing an E2F-1 cDNA, contained only four E2F-1 specific bands. Pretreatment of protein extracts, from both control and E2F-1 overexpressing cells, with lambda-phosphatase eliminated all E2F-1-specific bands except the single band migrating most rapidly on the gels. These data demonstrated that the multiple protein bands were differentially-phosphorylated forms of E2F-1 protein and showed that novel, more highly phosphorylated E2F-1 forms were present in cells overexpressing the E2F-1 protein. In addition, immunoprecipitation of the retinoblastoma (pRb) protein from cells overexpressing the E2F-1 cDNA showed that the novel, highly phosphorylated E2F-1 forms were preferentially coimmunoprecipitated, indicating that pRb bound preferentially to these E2F-1 forms.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , ADN Complementario/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Electroforesis en Gel de Poliacrilamida , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Fosforilación , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/aislamiento & purificación

RESUMEN

Overexpression of genes encoding E2F transcription factors can transform some cultured cell lines and cause apoptosis of others. Apoptosis due to E2F overexpression requires the presence of wild-type p53. Cell lines in which stable E2F overexpression is possible might be expected, therefore, to contain mutant p53. In this report, it was asked whether endogenous p53 was mutant or wild-type in four established fibroblast cell lines that this laboratory previously showed stably overexpressed and were transformed by exogenous E2F-1. Unexpectedly, it was found that the p53 in these cells was wild-type by the criteria of immunoprecipitation with conformation-specific, p53 monoclonal antibodies and by transactivation of a p53-dependent reporter gene construct in transient transfection assays. These data indicate that stable overexpression of E2F-1 is possible in the presence of wild-type p53 and may result in cell transformation.

Asunto(s)

Apoptosis/genética , Proteínas Portadoras , Proteínas de Ciclo Celular , Transformación Celular Neoplásica , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Células 3T3 , Animales , Línea Celular Transformada , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Técnicas de Transferencia de Gen , Ratones , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis

Asunto(s)

División Celular , Células Cultivadas , Células 3T3 , Animales , Adhesión Celular , Diferenciación Celular , Mamíferos , Ratones , Plásticos

RESUMEN

The E2F transcription factor can regulate expression of numerous cellular genes controlling proliferation, including proto-oncogenes and genes regulating cell cycle progression. Therefore, genes comprising the E2F gene family could potentially contribute to carcinogenesis. To test the potential of E2F to act as a transforming gene, a cDNA encoding E2F-1 was constitutively overexpressed in established rodent cells using a retroviral vector. Overexpressed E2F-1 was functional, as shown by stimulation of a transfected adenovirus E2 promoter driving a chloramphenicol acetyltransferase reporter gene in E2F-1 overexpressing cells. This stimulation was dependent on functional E2F binding sites in the promoter. Examination of phenotype showed that E2F-1 overexpression mediated cell transformation as measured by the ability of cells to form colonies in soft agar medium. In addition, overexpressed E2F-1 shortened the duration of the G1 cell cycle phase in proliferating cells, a property characteristic of other transforming genes. These data provide direct evidence that E2F-1 can act as a transforming gene and a critical regulator of cell cycle progression and suggest the possibility of E2F involvement in carcinogenesis.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN , Oncogenes/genética , Factores de Transcripción/genética , Activación Transcripcional , Células 3T3 , Animales , Sitios de Unión , Transformación Celular Neoplásica/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Fase G1/fisiología , Genes Reporteros , Vectores Genéticos , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Fenotipo , Regiones Promotoras Genéticas/fisiología , Proteína 1 de Unión a Retinoblastoma , Retroviridae/genética , Factor de Transcripción DP1 , Factores de Transcripción/biosíntesis , Transfección , Ensayo de Tumor de Célula Madre

RESUMEN

Few quantitative studies addressing immunofluorescence histogram analysis have been published. One study by Overton (Cytometry 9:619-626, 1988) has shown threshold and histogram subtraction methods to be accurate for analysis of well-separated immunofluorescence distributions of positive and negative cells. An evaluation of methods to analyze immunofluorescence histograms when positive and negative immunofluorescence distributions overlap has not, to our knowledge, been reported. In this paper, data obtained from flow cytometry of immunofluorescently stained cells infected with recombinant retroviruses that produce a range of simian virus 40 large T antigen levels were analyzed by threshold, histogram subtraction, and distribution modeling methods. This analysis showed that as the separation between the immunofluorescence distributions of positive and negative cell populations decrease the best methods for histogram analysis are modeling followed, in order, by histogram subtraction, and threshold analysis.

Asunto(s)

Antígenos Virales de Tumores/análisis , Separación Celular/métodos , Retroviridae/genética , Línea Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos

RESUMEN

Simian virus 40 (SV40) large T antigen (Tag) expression results in reduced percentages of G1-phase cells and increased percentages of S- and G2+M-phase cells in exponentially growing fibroblast populations as compared with identical cell populations not expressing Tag. This effect is the result of reduced G1 and increased G2+M cell cycle phase durations caused by Tag [Sladek, T.L. & Jacobberger, J.W. (1992). J. Virol., 66, 1059-1065]. Using recombinant retroviruses to manipulate Tag expression over a 25-fold range, it is shown here that the magnitude of this cell cycle phenotype increases as a function of increasing intracellular Tag concentration. This effect of Tag on the cell cycle is not independent of negative regulation by cellular mechanisms since exponentially growing cell populations producing high and increasing levels of Tag, increase the fraction of cells residing in G1 and decrease the fraction in S and G2+M as a function of cell density. Therefore, the data in this paper show, first, that Tag is a concentration-dependent, positive cell cycle regulator in exponentially proliferating cells and, second, that endogenous cellular mechanisms negatively regulating the cell cycle in response to cell density override the effect of Tag.

Asunto(s)

Antígenos Transformadores de Poliomavirus/fisiología , Ciclo Celular/genética , Regulación Viral de la Expresión Génica/fisiología , Ciclo Celular/inmunología , ADN Recombinante , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Fenotipo , Retroviridae , Transfección

RESUMEN

The effect of simian virus 40 large T-antigen (Tag) expression on the cell cycle of exponentially growing, established, mouse NIH 3T3 fibroblasts was examined by using a sensitive flow cytometric assay to analyze nonselected cells immediately after infection with a Tag-encoding recombinant retrovirus. Tag expression resulted in reduced percentages of G1-phase cells and increased percentages of S- and G2 + M-phase cells compared with cell populations infected with a control virus not encoding the Tag gene. Cell cycle-blocking drugs were used to examine the exit rate for each of the cell cycle phases, G1, S, and G2 + M, for Tag-expressing and Tag-nonexpressing cells growing in the same cell culture dish. As a result of Tag expression, the duration of the G1 phase was decreased (average G1-phase exit duration decreased by 18%) and the duration of the G2 + M phase was increased (average G2 + M exit duration increased by 29%). The duration of S phase was unaffected by Tag expression.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Ciclo Celular , Virus 40 de los Simios/fisiología , Células 3T3 , Animales , Ciclo Celular/efectos de los fármacos , División Celular , Fase G1 , Fase G2 , Virus Helper/genética , Virus Helper/fisiología , Cinética , Ratones , Mitosis , Nocodazol/farmacología , Virus 40 de los Simios/genética , Replicación Viral

RESUMEN

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.

Asunto(s)

Antígenos/análisis , Proteínas Bacterianas , Citometría de Flujo/métodos , Coloración y Etiquetado , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/análisis , Biotina , Ratones , Proteínas Recombinantes de Fusión , Sensibilidad y Especificidad , Estreptavidina

RESUMEN

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.

Asunto(s)

Antibacterianos/farmacología , Antígenos Transformadores de Poliomavirus/genética , Resistencia a Medicamentos/genética , Vectores Genéticos , Gentamicinas/farmacología , Retroviridae/genética , Virus 40 de los Simios/genética , Animales , Anticuerpos Monoclonales , Antígenos Transformadores de Poliomavirus/análisis , Ciclo Celular/efectos de los fármacos , Transformación Celular Viral , Células Cultivadas , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos , Virus 40 de los Simios/inmunología

RESUMEN

Transfection of Acholeplasma laidlawii strain JA1 with viral DNAs that have been sequence-specifically methylated in vitro has led us to previously postulate that JA1 cells contain an enzyme which cleaves only DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing that base. In this paper we show that an endonuclease activity is present in extracts from JA1 cells but not in extracts from a restriction-deficient variant of JA1. The partially purified enzyme cleaves not only DNA containing 5-methylcytosine, but also DNA containing no methylated bases. We discuss why this endonuclease activity may not have the same in vitro specificity as would be expected from in vivo experiments.

Asunto(s)

Acholeplasma laidlawii/enzimología , Proteínas Bacterianas/aislamiento & purificación , Citosina/análogos & derivados , Enzimas de Restricción del ADN/aislamiento & purificación , ADN Bacteriano/metabolismo , 5-Metilcitosina , Proteínas Bacterianas/metabolismo , Citosina/análisis , ADN/metabolismo , Enzimas de Restricción del ADN/metabolismo , ADN Circular/metabolismo , Especificidad por Sustrato

RESUMEN

We isolated two spontaneous variants of mycoplasma virus L2. Both variants, designated L2ins1 and L2ins2, contained a 3.1-kilobase-pair (kbp) insertion in the 11.8-kbp wild-type L2 genome. The insert DNA was shown to be derived from two noncontiguous regions of the L2 genome, and L2ins1 and L2ins2 differed only in the location of the 3.1-kbp insertion. We also isolated L2 miniviruses from serial passages of L2, L2ins1, and L2ins2 viruses. Miniviruses contained circular DNA molecules of 3.1 kbp or multimers of 3.1 kbp. Minivirus 3.1-kbp DNAs had the same sequences as the 3.1-kbp insert DNAs found in L2ins1 and L2ins2 viruses. Miniviruses were not infectious and interfered with the growth of L2, L2ins1, and L2ins2 viruses; hence, L2 miniviruses appeared to be defective interfering particles.

Asunto(s)

Bacteriófagos/genética , Genes Virales , Acholeplasma laidlawii , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Secuencia de Bases , ADN Circular/metabolismo , ADN Viral/metabolismo , Virus Defectuosos/genética , Virus Defectuosos/fisiología , Variación Genética , Mutación , Interferencia Viral

RESUMEN

Mycoplasmas are wall-less prokaryotes which have small genomes and are known to have evolved from ancestors of Gram-positive bacteria. A model is proposed to explain how mycoplasmas may have evolved from these ancestors which had cell walls and large genomes. It is proposed that the initial step in this process was loss of the cell wall and conversion of the ancestral bacterium to an L-form. Fusion of L-forms would have resulted in a single cell that contained two or more complete genomes. It is thought that this bringing together of multiple genomes by cell fusion resulted in genetic recombination between genomes and loss of DNA segments from the cell. Data from bacterial systems are cited in support of this model.

Asunto(s)

Evolución Biológica , Mycoplasma/genética , Genes Bacterianos , Modelos Genéticos , Recombinación Genética

RESUMEN

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.

Asunto(s)

Acholeplasma laidlawii/genética , Citosina/análogos & derivados , ADN Bacteriano/análisis , 5-Metilcitosina , Bacteriófagos/genética , Secuencia de Bases , Deleción Cromosómica , Citosina/análisis , Enzimas de Restricción del ADN , ADN Bacteriano/metabolismo , Metilación , Mutación , Transfección

RESUMEN

We have found that mycoplasma virus L172 is an enveloped globular virion containing circular, single-stranded DNA of 14.0 kilobases. L172 has been reported by other workers to have a double-stranded DNA genome of 13 to 17 kilobase pairs and has been classified as a plasmavirus, a group for which mycoplasma virus L2 is the type member. Mycoplasma viruses L172 and L2 differ in genome size and structure, DNA base composition, and protein composition, and they have no detectable DNA homology. As the only reported enveloped virion containing single-stranded DNA, L172 represents a new group of viruses.

Asunto(s)

Bacteriófagos/genética , ADN Circular/análisis , ADN de Cadena Simple/análisis , ADN Viral/análisis , Genes Virales , Acholeplasma laidlawii , Bacteriófagos/análisis , Bacteriófagos/clasificación , Bacteriófagos/crecimiento & desarrollo , Composición de Base , Enzimas de Restricción del ADN , Hibridación de Ácido Nucleico , Proteínas Virales/análisis , Virión/ultraestructura

RESUMEN

Double-stranded DNA from mycoplasma virus L2 can transfect Acholeplasma laidlawii cells in the presence of polyethylene glycol (T. L. Sladek and J. Maniloff, J. Bacteriol. 155:734-741, 1983). We report here that both single-stranded DNA and double-stranded replicative form DNA, from the single-stranded DNA mycoplasma virus L51, are also infectious in this system. For both DNAs transfection frequencies were in the range of 10(-8) transfectants per DNA molecule and 10(-3) transfectants per CFU. An unexpected finding was that both DNAs could transfect A. laidlawii strain REP-, a variant which is a nonpermissive host for single-stranded DNA mycoplasma viruses due to a block in viral DNA replication (Nowak et al., J. Bacteriol. 127:832-836, 1976). The number of viruses produced by transfected REP- cells was comparable to the number produced by both transfected and infected wild-type cells. Therefore, transfected L51 DNAs are able to bypass the replication block in REP- cells that occurs when these cells are infected by L51 virions.

Asunto(s)

Acholeplasma laidlawii/genética , Bacteriófagos/genética , ADN de Cadena Simple/genética , ADN Viral/genética , Mycoplasma/genética , Transfección , ADN de Cadena Simple/aislamiento & purificación , ADN Viral/aislamiento & purificación , Cinética , Especificidad de la Especie