The incorporation of thymidine into deoxyribonucleic acid in mouse fibroblasts infected with polyoma virus.
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ABSTRACT:
SUBMITTER: Molteni P
PROVIDER: S-EPMC1264797 | biostudies-other | 1966 Jan
REPOSITORIES: biostudies-other
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Project description:To develop a new form of DNA coupling under mild reaction and coupling conditions, DNA oligonucleotides were synthesized containing a 3' ribonucleotide. Upon reaction with millimolar sodium metaperiodate (NaIO4), the ribose is oxidized to a dialdehyde at pH 6.8. This reaction is complete in 30min, is quenched with millimolar sodium metabisulfite (Na2S2O5) and is then suitable for coupling to hydrazide-agarose supports. Coupling occurs with a half-time of 27min and 80% couples in 2h. The EP18 oligonucleotide which binds to the CAAT enhancer binding protein (C/EBP) was synthesized with a 3' ribose (rEP18) and coupled to hydrazide-agarose. The columns prepared show no significant loss of the oligonucleotide after 50 days. A crude bacterial extract from cells expressing a chimeric fusion protein of GFP-C/EBP was applied to the columns and eluted with different salt concentrations. The active protein elutes in 0.5M NaCl and SDS-PAGE/silver stained gels show a single major band which comigrates with GFP-C/EBP as well as three minor contaminants. This provides a new alternative way of coupling DNA to solid supports using mild chemistry which is non-detrimental to the DNA and can be performed if required in the presence of nuclear extract.
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Project description:DNA-dependent RNA polymerase (EC 2.7.7.6) ACTIVITIES FROM NORMAL BHK-21/C13 cells and from BHK-21/C13 cells transformed by polyoma virus (PYY cells) were solubilized and fractionated on columns of DEAE-Sephadex. Various properties of the A and B enzymes from the two types of cell were compared. 1. The yields of polymerase relative to the DNA content of the nuclear preparations are similar for both cell types. 2. The ionic-strength optima of polymerases A and B are 12.5 mM and 100mM with respect to (NH4)2SO4 for both cell types. 3. The Mn2+/Mg2+ activity ratio (measured at the respective optimum for each cation) for polymerase A from BHK-21/C13 cells was 1.48 and for the polymerase A from PYY cells was 0.55. The corresponding ratios for polymerase B were 10.11 for BHK-21/C13 cells and 22.75 for PYY cells. 4. Minor differences in the ability of the A polymerases to transcribe native and denatured DNA templates were observed; such differences were not apparent when the B polymerases were compared. 5. All the polymerases were inhibited completely by actinomycin D and by rifampicin AF/013, but not markedly so by rifampicin. Alpha-amanitin inhibited polymerase B but not polymerase A.
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Project description:Neutrophils are derived from bone marrow hematopoietic stem cells (HSCs) and are the largest population among circulating white blood cells in humans, acting as the first line of defense against invading pathogens. Whether neutrophils can be generated by transdifferentiation strategies is unknown. Here, we show that thymidine induces the conversion of mouse fibroblasts to neutrophils. Induced neutrophils (iNeus) showed antibacterial effects and did not undergo malignant transformation in vivo. Importantly, iNeu transplantation cured neutropenia in mice in vivo. Mechanistically, thymidine mediates iNeu conversion by enhancing Tet3 activity. Tet3 initiates the expression of the neutrophil fate decision factors Cebpδ and Rfx1 that drive the transdifferentiation of mouse fibroblasts to neutrophils. Therefore, the induction of functional neutrophils by chemicals may provide a potential therapeutic strategy for patients with neutropenia patients and infectious diseases.Fibroblasts; Neutrophils; Thymidine; Transdifferentiation; Tet3.
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Project description:MA/MyJ mice express a natural antibody to the highly oncogenic polyoma virus. C57BR/cdJ mice lack this antibody but mount an adaptive T-cell response to the virus. Analysis of F2 progeny of a cross between these strains reveals a pattern of inheritance of expression of the natural antibody involving two genes in an epistatic relationship.
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Project description:The extent of phosphorylation of ribosomal protein S6 was compared in normal hamster fibroblasts and in fibroblasts transformed by polyoma virus or simian virus 40. In both strains of transformed cells the protein was more highly phosphorylated than in the normal cells.
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Project description:BackgroundPolyoma virus-associated nephropathy (PVN) is a common cause of renal transplant failure. The risk factors for the development of PVN have not yet been studied in large cohorts of patients for periods of 20 years.MethodsWe collected clinical, renal biopsy and urinary cytology data from all patients with renal transplantations performed at the University Hospital of Basel from 1985 to 2005. All patients with a renal biopsy and urine cytology were included (n = 880). Renal transplants were divided into three groups, according to evidence of polyoma virus (PV) infection (decoy cells in the urine) and biopsy-proven PVN: Renal transplants without evidence of a PV infection (n = 751). Renal transplants with PV reactivation, e.g. decoy cell (DC) found by urinary cytology, but without PVN (n = 90). Renal transplants with PVN (n = 39).ResultsThe prevalence of biopsy-proven PVN in this cohort of patients was 3.3%. Immunosuppression with mycophenolate and/or tacrolimus, ATGAM, male gender of the recipient and a higher number of transplant rejection episodes were factors significantly associated with PVN development.ConclusionsThe most important risk factors for the development of PVN are acute rejection and ATGAM used as induction therapy as well as tacrolimus and mycophenolate as maintenance therapy. Therefore, we conclude that patients with tacrolimus and mycophenolate maintenance therapy should be carefully monitored for the development of PVN.
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Project description:INTRODUCTION:Replication of the human immunodeficiency virus involves an obligatory step of reverse transcription of the viral ribonucleic acid genome into a double-stranded deoxyribonucleic acid, and subsequent integration of the deoxyribonucleic acid into the human chromatin to form the proviral deoxyribonucleic acid. This proviral human immunodeficiency virus deoxyribonucleic acid is a critical marker for the diagnosis of acute infections, mother-to-child transmissions and for the confirmation of indeterminate serological reactions. We describe a case of a human immunodeficiency virus positive woman, naïve to antiretroviral treatment, who was persistently negative for human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction. This observation, to the best of our knowledge, is the first time that it has been described in Africa. CASE PRESENTATION:A 28-year-old Gabonese woman living in Cameroon requested a human immunodeficiency virus diagnosis in our laboratory. She had an unprotected heterosexual contact 6 months earlier while on vacation in Gabon. The request for a human immunodeficiency virus test was as a result of apprehensions developed after the exposure episode. Human immunodeficiency virus serological examinations were ambiguous and confirmatory tests (including human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction) were carried out. Apart from the human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction that was persistently negative, all other polymerase chain reactions carried out were positive. The deoxyribonucleic acid sequences have been submitted to the GenBank database with accession numbers: KC626022, KC626023 and KC626024 for the protease, reverse transcriptase and gp41 genes respectively. CONCLUSION:The persistently negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction in a person with a confirmed human immunodeficiency virus infection is of immense importance in the human immunodeficiency virus diagnostic field. This could highlight the fact that cases of false negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reactions exist especially with the high genetic variations observed with human immunodeficiency virus. The challenges presented by such false negative tests in the identification of acute infections, mother-to-child transmissions and the confirmation of indeterminate serological reactions are daunting. These data therefore would be invaluable especially to clinicians in Africa where non-B human immunodeficiency virus subtypes circulate.
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Project description:1. DNA has been isolated in 90% yield from T5-infected cultures of Escherichia coli ;pulse'-labelled with [(3)H]thymidine. It had a buoyant density in caesium chloride solution identical with the DNA of mature T5 phage, and no components of unusual buoyant density were detected. 2. The DNA preparation was resolved into two major components of differing specific activity on a column of kieselguhr coated with methylated serum albumin. The DNA of high specific activity could be eluted from the column only with 2n-ammonia, and the firm binding did not appear to be due to an artifact of preparation. 3. A similar fractionation into two DNA components of differing specific activity was observed when the ;pulse'-labelled culture was lysed with sodium dodecyl sulphate and the lysate rocked with phenol. The DNA of high specific activity was found in the interface precipitate between the phenol and aqueous layers. 4. The amounts of DNA in the two fractions were measured at different times after infection and the radioactivity content of each was determined at various times after a short ;pulse' of [(3)H]thymidine. The interface fraction contained the replicating phage DNA, and the DNA from mature phage particles appeared in the aqueous fraction. 5. Analogous results were obtained with T2-infected E. coli. In the presence of chloramphenicol the DNA in the interface fraction was not converted into DNA extractable into the aqueous layer. Since chloramphenicol prevents the condensation of DNA into phage heads, it is suggested that any DNA in extended configuration is trapped inside the rigid-layer framework of the cell wall. 6. Treatment with lysozyme released much of the DNA from the interface precipitate. This DNA was firmly bound by the chromatographic column and had the same buoyant density in caesium chloride solution as normal T5-phage DNA. Sucrose-gradient sedimentation studies showed that it was heterogeneous and that as much as 60% sedimented faster than T5-phage DNA.
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Project description:Aberrant methylation has been regarded as a hallmark of cancer. 5-hydroxymethylcytosine (5hmC) is recently identified as the ten-eleven translocase (ten-eleven translocase)-mediated oxidized form of 5-methylcytosine, which plays a substantial role in DNA demethylation. Cell-free DNA has been introduced as a promising tool in the liquid biopsy of cancer. There are increasing evidence indicating that 5hmC in cell-free DNA play an active role during carcinogenesis. However, it remains unclear whether 5hmC could surpass classical markers in cancer detection, treatment, and prognosis. Here, we systematically reviewed the recent advances in the clinic and basic research of DNA 5-hydroxymethylation in cancer, especially in cell-free DNA. We further discuss the mechanisms underlying aberrant 5hmC patterns and carcinogenesis. Synergistically, 5-hydroxymethylation may act as a promising biomarker, unleashing great potential in early cancer detection, prognosis, and therapeutic strategies in precision oncology.
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