Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

AT least four established human lymphocyte cell lines, one that originates from a Burkitt's lymphoma and the others from normal persons, contain Epstein-Barr virus (EBV) genome¹. These cells show no viral antigens by immunofluorescence tests nor do they produce virus particles. We are examining one of the four cell lines, Raji (cells from a Burkitt's lymphoma), in more detail. The DNA isolated from purified Raji chromosomes contains as much virus genome as the DNA extracted from whole cells (65 genome equivalents per cell)¹. The viral DNA therefore seems to be in the chromosomes. This result, however, does not necessarily indicate that the viral DNA is physically integrated into chromosomal DNA. The following experiments suggest that the EBV DNA in Raji cells is not covalently linked to the large chromosomal DNA, although the number of viral genomes per cell remains constant during passage. The results do not, however, exclude the possibility that small fragments of cell DNA are bonded to the viral DNA. The data also indicate that EBV DNA in Raji cells exists in strands of complete or nearly complete size.     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl₂, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From ³²P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Iron salts and transferrin are specifically required for cell division of cultured 3T6 cells.

3T6 Swiss mouse fibroblasts can be plated in medium without serum. Prostaglandin F₂^∞, fibroblastic growth factor, epidermal growth factor and insulin stimulate DNA synthesis in medium containing vitamin B₁₂. A combination of these factors, however, does not stimulate cell division under our conditions. Iron salts and transferrin or low concentrations of serum are required to be concurrently present with the growth factors before cell division is observed.     

DNA methylation of cord blood cell types: Applications for mixed cell birth studies

Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test P < 10^?8). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at P < 10^?8) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.     

CLEARANCE RATE OF EXOGENOUS 3H-THYMIDINE FROM THE PLASMA OF PREGNANT RHESUS MONKEYS

The plasma clearance rate of ³H-TdR was determined by thin-layer chromatography of samples from pregnant rhesus monkeys who had received a single intravenous injection of ³H-TdR. At 10 min after the injection the circulating levels of ³H-TdR had fallen to less than 0.2% of the initial theoretical distribution of the label. In general the plasma clearance rate can be described by a ‘double exponential’curve with half-lives of 1 min and 20 min. Thus, ³H-TdR disappears from the circulation of the pregnant monkey more rapidly than it does from the bloodstream of rodents, which correlates with previously detected interspecies differences in autoradiographic labeling of neurons. This means (1) that higher doses of ³H-TdR must be used for autoradiographic labeling of monkey tissues and (2) that the monkey may be useful for studying cell kinetics because it has a short pulse labeling of the DNA.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.     

Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P_PC1 = 1.4 × 10^?9; P_PC2 = 2.9 × 10^?5; P_PC3 = 3.8 × 10^-5; P_PC4 = 4.2 × 10^-6; P_PC5 = 9.9 × 10^-13, P_PC6 = 1.3 × 10^?11) and not with sample type (P_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P<10^?5). Estimated cell type proportions did not differ by sample type (P = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.     

Inhibition of 3H-thymidine incorporation of lymphocytes by a soluble factor from macrophages

Supernatants of peritoneal macrophages cocultivated with spleen cells or thymocytes contain a factor suppressing the ³H-thymidine (³H-TdR) incorporation of PHA-stimulated lymphocytes. The suppressing activity is not due to cytotoxicity and does not affect the rate of cell transformation or cell proliferation. The factor reduces only the ³H-TdR incorporation and not the DNA synthesis of lymphocytes. It does not show target cell specificity. The factor is dialysable, heat stable, and generated by macrophages.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.     

A Requirement for DNA Synthesis during Auxin Induction of Cell Enlargement in Tobacco Pith Tissue

IAA (indoleacetic acid) is known to induce cell enlargement without cell division in tobacco pith explants grown on an agar medium without added cytokinin. The very long lag period before IAA (2 × 10^?5M) stimulates growth, about 3 days, can be useful to study the metabolic changes which lead to the promotion of growth. When the disks are transferred to a medium without IAA after 2 days or less of treatment with IAA, the IAA does not stimulate growth. Disks transferred after 3 days, subsequently show an auxin response, almost as great as those given IAA continuously. At 5 × 10^?4M, 5-fluorodeoxyuridine (FUDR), which inhibits DNA synthesis by blocking formation of thymidylate, completely suppresses the lAA-induced growth if it is added together with the IAA or 1 day later. When the FUDR is given 2 days after the IAA, there is a small increment of auxin-induced growth, and an even greater amount if added after 3 days. The period when exogenous auxin must be present to stimulate growth corresponds to the period of FUDR sensitivity. The FUDR inhibition is prevented by thymidine but not by uridine. Other inhibitors of DNA synthesis, hydroxyurea and fluorouracil, also inhibit auxin-induced growth. Thus DNA synthesis seems to be required for auxin induction of cell enlargement in tobacco pith explants. In contrast, FUDR does not inhibit auxin-induced growth in corn coleoptile and artichoke tuber sections.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

A rapid and sensitive assay for pyrimidine dimers in DNA

We have developed a rapid, sensitive assay for pyrimidine dimers. The assay has greatly facilitated the purification and characterization of the photoreactivating enzyme. The procedure depends on (1) the resistance of the nucleotide phosphate bond in dimer-containing regions of DNA to attack by DNase I, venom phosphodiesterase and alkaline phosphatase and (2) selective adsorption to Norit of mononucleosides and ³²P-labeled, dimer containing oligonucleotides (but not ³²P₁) resulting from nuclease digestion of highly-purified, ³²P-labeled bacteriophage DNA. The method is sensitive and rapid. The presence of the usual nuclease activities found in cell extracts does not interfere with the assay. Thus photoreactivating enzyme activity can be detected even in the presence of non-specific or uv-specific nucleases. Neither photoreactivation nor the digestion reaction is affected by purification agents at concentrations commonly used in enzyme purification.     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph^p mutants reveals an acceleration of S phase, showing that ph^p regulates S phase length. Like ph^p mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph^p are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.     

Cellular and tissue responses to heavy ions: Basic considerations

Summary Responses of the S/S variant of the L5178Y murine leukemic lymphoblast, the photoreceptor cell of the rabbit retina and the lenticular epithelium of the rabbit to heavy ions (²⁰Ne,²⁸Si,⁴⁰Ar and⁵⁶Fe) are described and discussed primarily from the standpoint of the need for a comprehensive theory of cellular radiosensitivity from which a general theory of tissue radiosensitivity can be constructed.The radiation responses of the very radiosensitive, repair-deficient S/S variant during the G₁- and early S phases of the cell cycle were found to be unlike those of normally radioresistant cells in culture: the relative biological effectiveness (RBE) did not increase with the linear energy transfer (LET) of the incident radiation. Such behavior could be anticipated for a cell which is lacking the repair system that operates in other (normal) cells when they are exposed to ionizing radiations in the G₁ phase of the cell cycle. The S/S variant does exhibit a peak of radioresistance to X-photons mid-G₁ + 8 h into the cell cycle, however, and as the LET was increased, the repair capacity responsible for that radioresistance was reduced progressively.Sensory cells (photoreceptors) in the retina of the New Zealand white (NZW) rabbit are very radioresistant to ionizing radiations, and several years elapsed after localized exposure (e.g., 5–10 Gy) to heavy ions (²⁰Ne,⁴⁰Ar) before photoreceptor cells were lost from the retina. During the first few weeks after such irradiations, damage to DNA in the photoreceptor cells was repaired to a point where it could not be demonstrated by reorienting gradient sedimentation under alkaline conditions, a technique that can detect DNA damage produced by <0.1 Gy of X-photons. Restitution of DNA structure was not permanent, however, and months or years later, butbefore loss of photoreceptor cells from the retina could be detected, progressive deterioration of the DNA structure began.