Number Of Genome Equivalents Research Articles

Comparison of polymerase chain reaction and flow cytometry for measuring telomere length of human leukocytes.

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.

Occurrence of Cronobacter Spp. in Ready-to-Eat Vegetable Products, Frozen Vegetables, and Sprouts Examined Using Cultivation and Real-Time PCR Methods.

Environmental matrices and food products are hypothesized to be sources of Cronobacter spp. The severity of neonatal infections, increasing number of cases in elderly and immunocompromised individuals, as well as isolation of Cronobacter spp. from clinical materials demands that more attention should be paid to Cronobacter spp. detection and occurrence of the bacteria in food products. Here, a total of 175 samples of ready-to-eat vegetables, frozen vegetables, and sprouted seeds were collected during a period of 1 year and examined for the presence of Cronobacter spp. using a cultivation method with two different sample preparations and real-time polymerase chain reaction (qPCR). In total, Cronobacterspp. were detected in 22.3% of tested samples using cultivation. In comparison, direct qPCR detected Cronobacterspp. in 37.7% of these samples (p < 0.01; Fisher's exact test) and the numbers of genome equivalents per gram reached 108 in some samples of sprouts. Cronobacter spp. were isolated from 51.4%, 37.2%, and 5.2% samples of sprouts, frozen vegetables, and cut green leaves/salads, respectively. Using qPCR, the most frequently contaminated sample types were sprouts (91.4%) and frozen vegetables (60.5%), whereas the rate of positivity for cut green leaves/salads was, in comparison, only 8.2% (p < 0.01; χ2 -test for independence). PRACTICAL APPLICATION: This study provided valuable information on the occurrence of Cronobacter spp. in ready-to-eat vegetables using cultivation and qPCR. Cronobacter spp. are emerging opportunistic pathogens that can be present in food of plant origin. Cronobacter spp. were isolated from sprouts, frozen vegetables, and cut green leaves/salads, and the numbers of genome equivalents per gram reached 108 in some samples of sprouts.

Development of pulmonary chlamydia infection in inbred mice strains differentiated by genetically determined sensitivity to tuberculosis infection

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. Susceptibility does not depend upon the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by a few interacting QTL mapped to chromosomes 3, 9 and 17. To find out whether tuberculosissusceptible I/St mice are susceptible to other intracellular bacteria, we investigated taxonomically distant pathogen, Chlamydia pneumoniae. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former are more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St 9.2 ± 1.2 days, A/Sn − 22.0 ± 2.0 days (p < 0.001). To estimate the degree of chlamydial multiplication in the lungs, we established a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite’s genome equivalents in infected tissue from 1 to 16 days after challenge. Interstrain difference of chlamydia burden in lungs we obtained after 24 hours after infection only. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection, and the numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-α and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

Plasma cell‐free DNA in ovarian cancer

Cell-free DNA reflects both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. The authors sought to determine the role of preoperative total plasma cell-free DNA levels in predicting clinical outcome in patients with ovarian cancer. After institutional review board consent, DNA was extracted from plasma of 164 women with invasive epithelial ovarian carcinoma (EOC), 49 with benign ovarian neoplasms, and 75 age-matched controls. The samples were randomly divided into training (n = 144) and validation (n = 144) sets. Quantification of cell-free DNA was performed using real-time polymerase chain reaction for beta-globin, and the number of genome equivalents (GE) per milliliter of plasma was determined. Cell-free DNA was correlated with clinicopathologic parameters. The training and validation sets were similar in terms of demographic features. In the training set, EOC patients had a median preoperative cell-free DNA level of 10,113 GE/mL, compared with patients with benign ovarian neoplasms (median, 2365 GE/mL; P < .0001) and controls (median, 1912 GE/mL, P < .0001). Cell-free DNA >22,000 GE/mL was significantly associated with decreased patient survival (P < .001). After adjusting for other clinical variables, preoperative cell-free DNA >22,000 GE/mL was an independent predictor (P = .02) for disease-specific survival. Analysis of the validation set confirmed significantly higher cell-free DNA levels in EOC (median, 13,672 GE/mL) and that cell-free DNA >22,000 GE/mL was associated with a 2.83-fold increased risk of death from disease (P < .001). Preoperative plasma total cell-free DNA levels are significantly elevated in patients with EOC. Elevated plasma cell-free DNA is an independent predictor for death from disease in ovarian cancer.

Circulating cell-free DNA: A novel biomarker for response to therapy in ovarian carcinoma

Introduction: Cell-free DNA (CFDNA) is a reflection of both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. We sought to determine whether tumor-specific plasma DNA could be used as a biomarker for tumor burden and response to therapy in an orthotopic ovarian cancer model. Methods: Female nude mice injected intraperitoneally with HeyA8 ovarian cancer cells were treated with either docetaxel alone or in combination with anti-angiogenic agents (AEE788 -- dual VEGFR and EGFR antagonist or EA5 – monoclonal antibody against ephrin A2). Following DNA extraction from plasma, quantification of tumor-specific DNA was performed by real-time PCR using human specific beta-actin primers. The number of genome equivalents (GE/ml) were determined from a standard curve. Apoptosis was assessed by TUNEL staining of treated tumors. Results: The levels of tumor-specific DNA in plasma increased progressively with increasing tumor burden (R2=0.8, p

Quantification of Total Plasma Cell‐Free DNA in Ovarian Cancer Using Real‐Time PCR

Our objective was to compare the levels of total circulating plasma cell-free DNA (CfDNA) using real-time PCR in patients with late-stage ovarian cancer with those in unaffected controls. Following IRB consent, DNA was extracted from archived frozen plasma of 19 patients with primary ovarian carcinoma and 12 age-matched controls using Qiagen DNA Isolation Kits. Quantification of total CfDNA was performed using real-time PCR with the TaqMan Assay for GAPDH, beta-actin and beta-globin and the number of genome equivalents (GE/mL) were determined from a standard curve. CfDNA levels of these loci were compared between the groups with Student's t-test, with P < 0.05 being statistically significant. The mean age of the patients was 61.6 years (+/-9.6) and of the controls was 54 years (+/-12.2). All patients had high-grade, advanced stage (III or IV) serous ovarian carcinomas. Preoperative CA-125 levels ranged from 43 to 15,626 IU/mL (mean 2487.2 +/- 3686 IU/mL). Total CfDNA in ovarian cancer was higher among patients with ovarian cancer as compared to controls at all three loci: GAPDH (P = 0.022), beta-actin (P = 0.025), and beta-globin (P = 0.0089). CfDNA is elevated in advanced stage disease compared to controls. These preliminary results suggest that total CfDNA in the plasma of patients with ovarian cancer may be useful for noninvasive screening and disease surveillance.

QUANTITATIVE PCR DATA FALSIFY THE CHROMOSOMAL ENDOREDUPLICATION HYPOTHESIS FOR VOLVOX CARTERI (VOLVOCALES, CHLOROPHYTA)

Two conflicting hypotheses for chromosome replication in the Volvocaceae, one postulating multiple rounds of replication prior to cell division (endoreduplication) and the other claiming a canonical sequence of one round of nuclear DNA replication preceding each cell division, have been tested experimentally. Competitive PCR of the single‐copy actin gene (target) of Volvox carteri f. nagariensis Iyengar and a shortened gene version (competitor) containing the same primer binding sites were used to assess the genome equivalents present in a given number of cells. Determining the molar ratio of the PCR products generated from target DNA (extracted from a known number of cells) and defined numbers of competitor molecules revealed that Volvox embryos between the one‐ and 16‐cell stages possess an average of between one and two—but never more than two—copies of the actin gene. This led us to conclude that the number of genome equivalents per nucleus in dividing Volvox embryos varies only between one and two and that, unlike the case predicted by endoreduplication, the nuclear genome undergoes only one round of replication prior to each cell division.

Comparative quantification of human cytomegalovirus DNA in blood of immunocompromised patients by PCR and Murex Hybrid Capture™ System

Background: Monitoring of human cytomegalovirus (HCMV) load by quantification of antigenemia, viremia and DNAemia is helpful in the management of HCMV infections in immunocompromised patients. In fact, threshold values of these viral parameters are associated with the emergence of clinical symptoms. In addition, the response to antiviral treatment is revealed by a decrease in viral load or virus disappearance from blood. Objectives: Aim of this study was to compare HCMV DNA quantification in blood of immunocompromised patients by an `in house' developed quantitative PCR (Q-PCR) assay and the commercially available Murex Hybrid Capture™ System (HCS). Study design: HCMV DNA was quantified in 95 blood samples from 12 heart and heart-lung transplant recipients and 27 AIDS patients using both techniques. For HCS analysis 3.5 ml whole blood were utilized, whereas Q-PCR was performed using 1×10 5 peripheral blood leukocytes (PBL). HCMV DNA levels obtained by HCS and Q-PCR were expressed as number of genome equivalents (GE)/ml whole blood or 1×10 5 PBL, respectively. Results from HCS and Q-PCR were compared and submitted to statistical analysis. In addition, HCMV DNA values were compared to levels of antigenemia and viremia. Results and Conclusions: Sensitivity of HCS, antigenemia and viremia with respect to Q-PCR were 37.2, 79.5 and 33.3%, respectively. Specificity was 100% for all techniques. On average, samples positive by Q-PCR only, contained low amounts of HCMV DNA. In particular, 45 (91.8%) out of 49 samples negative by HCS and positive by Q-PCR showed <500 GE/1×10 5 PBL. A significant correlation was found between quantitative DNA levels in samples positive by both HCS and Q-PCR ( n=29, R=0.693, P<0.01). HCS positivity was associated to significantly higher DNA values as determined by Q-PCR as well as to significantly higher antigenemia and viremia levels. A decrease in DNAemia levels was observed using both HCS and Q-PCR after antiviral treatment. Given that the great majority of blood samples missed by HCS contain low levels of HCMV DNA which are not clinically significant, HCS seems very promising as an alternative to HCMV DNA quantification by PCR in solid organ transplant recipients and AIDS patients.

The effect of growth rate of Micrococcus radiodurans on its heat sensitivity.

The growth rate of Micrococcus radiodurans R1 depends on the composition of its media. It grows faster in a rich medium than in a poor medium. However, there was no difference in heat sensitivity among the cells grown in media whose compositions were different. Recently, HANSEN reported that the number of genome equivalents of DNA per call of this bacterium changed from approiximately 4 to 10 depending on the growth rates in different media and that recombination repair between intact homologous segments of multiple genomes played an imporant role in its extremely high radioresistance. However, our data indicate that the growth rate of this organism does not influence the heat sensitivity. Therefore, it is concluded here that recombination repair for DNA damage by heat is scarcely influenced by multiple genomes in a cell of M. radiodurans.

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains.

We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep+ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep+ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep+ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.

Analysis of mouse embryonic gene library for the frequency of single and multiple copy genes.

A gene library was constructed from embryonic mouse DNA by ligating DNA fragments generated by partial Eco RI digestion with Charon 4A vector and in vitro packaging. A special consideration was given to randomization of target DNA. The general applicability of a gene library prepared in this manner was assessed through cloning a variety of genes of known reiteration frequency in the mouse genome. The survey included a single copy gene--C region of the immunoglobulin heavy chain, and genes that appear in more than one copy--V region of the immunoglobulin light chain genes and the endogenous retrovirus related genes. In all cases tested the frequency of clone isolation was in good agreement with the expected incidence based on the number of genome equivalents screened and the reiteration frequency of that particular gene. Moreover, we found no preference with regard to the clonability of genes contained in fragments of a wide-size range.

The biology of EB virus

The target cell most commonly infected by the ubiquitous EB virus is the B lymphocyte, and infection <i>in vitro</i> results in transformation and establishment of lymphoblastoid cell lines. Following transformation the EBV genome exists in both an integrated and an episomal form, and the number of genome equivalents per cell shows considerable variation. Transformed cells vary also in the level of expression of infectious virus, and may exhibit a number of EBV-related antigens detectable by immunofluorescence (IF), complement fixation, immunodiffusion or radioimmunoassay, The viral capsid antigen (VCA) is useful in routine diagnostic IF tests for antibody to EBV. The presence of an EBV-associated nuclear antigen (EBNA) correlates well with the detection in the cells of EBV genome bk molecular hybridization and EBNA is therefore a relatively simple indicator of the EBV-transformed state of cells. There is also a complex of membrane antigens (MA) on the surface of transformed cells. In addition to this transformation of normal B lymphocytes. EBV is able to superinfect lymphoid cell lines of B cell origin but are devoid of EBV genome. Finally, there is some evidence that certain epitheloid cell lines may possess EBV receptors. Three clinical entities have been so far associated with EBV; infectious mononucleosis (IM), Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPL). In IM, it is generally accepted that the atypical mononuclear cells represenr largely a response of T lymphocytes to EBV-infection of B lymphocytes. Antibodies to EBV usually appear by the time of onset and a rise in titre to VCA after onset is rarely detected. Transient IgM antibody to EBV occurs and provides a more specific serological test. In convalescence and thereafter. EBV-infected cells are found consistently only in lymph nodes, unless recrudescence occurs. The association of BL with EBV initially rested on the increased antibody response to EBV and received support by the detcction of the EBV genome in most BL of African origin although the non-African form may or may not carry the genome. A problem in the biology of EBV is the form of the virus during its long persistence following the primary infection. Our <i>in vitro</i> studies have alloued the development of an experimental system in which typical EBV transformation is completely inhibited. The inhibited lymphocytes are infected and carry the EBV genome, and the inhibition is reversible by simple adjustment of the culture conditions. Inhibition of EBV transformation is specifically mediated by a mononuclear cell population from the peripheral blood of persons with prior EBV infection. The importance of these findings is the demonstration that an immune process can modify EBV infection of lymphocytes without cell death, and prevent their subsequent proliferation as transformed cells.

Regulation of the synthesis of the lactose repressor.

Measurements of the lactose repressor over a tenfold range of cell growth rates were made on protein extracts from Escherichia coli cultures grown in media with various carbon energy sources. The concentration of lactose repressor varied with the number of genome equivalents per cell over this range in growth rates, suggesting that the number of lactose molecules within the cell is determined by the number of I gene copies present. The timing of repressor synthesis during the cell division cycle and its correlation with deoxyribonucleic acid synthesis was examined by synchronizing the cell division cycle of E. coli ED1039, in which the Lac region has been transposed from 10 to 36 min on the genetic map. Measurements of lactose repressor in the synchronized culture revealed a burst of repressor synthesis at the time of I gene duplication. The concentration of lactose repressor was found to decrease as a function of total cell protein during the division cycle until an increase in synthesis occurred, suggesting that repressor synthesis probably does not occur throughout the division cycle. A model for I gene regulation is proposed.