Amount Of Deoxyribonucleic Acid Research Articles

Étude comparative de l'adsorption d'acide déoxyribonucléique sur le chrysotile et le chrysotile phosphorylé (chrysophosphate)

Deoxyribonucleic acid (DNA) adsorption on one sample of Canadian chrysotile and two samples of chrysophosphate has been studied using chemical analysis and a surface method of analysis (photoelectron spectroscopy, XPS). Chrysophosphate was obtained using a phosphorylation process applied to chrysotile fibres; one chrysophosphate sample was opened in a Pallmann device. XPS analysis of the samples before DNA adsorption has shown that some surface phosphorus was dissolved following suspension of the fibres in distilled water. After DNA treatment, the amount of DNA adsorbed at the surface of the chrysophosphate sample was lower than that adsorbed at the chrysotile surface (12.6 ± 1.7 μg/mg and 74.0 ± 4.0 μg/mg respectively). In addition, the opened sample adsorbed an intermediary DNA amount (28.3 ± 5.6 μg/mg), suggesting that the phosphorylation process has not reached all the bulk fibres. The results indicate that DNA adsorption is a sensitive method for investigation of the surface state of the fibre. Moreover, it seems that the phosphorylation process masks or blocks the reactive surface sites for DNA adsorption.

7] Random cloning and sequencing by the M13/dideoxynucleotide chain termination method

This chapter discusses random cloning and sequencing by the M13/dideoxynucleotide chain termination method. The dideoxy chain terminator/Ml3 vector method of deoxyribonucleic acid (DNA) sequencing is considered to be the fastest method to determine the sequence of large fragments of DNA. Lengths of DNA are cloned into the bacteriophage M13 that provides a source of large quantities of single-stranded DNA. This single-stranded DNA can then be used as a template in a primer extension dideoxynucleotide sequence reaction. The chapter discusses the problems related to the simplest method of breaking a DNA molecule into subfragments, which uses restriction endonucleases. The easiest method of purifying the fragment to be sequenced from the cloning vector is cleavage with suitable restriction endonuclease(s) followed by fractionation of the restricted DNA and isolation from a low gelling-temperature agarose minigel. The chapter discusses several procedures, such as isolation of fragment, fragment self-ligation, sonication, and others. Regarding size selection for DNA purification, two factors determine the minimum size to be selected. First, the insert to be sequenced should be at least as long as the maximum, which can be read from a normal sequence gel run. Second, as the subfragments have ends generated at random and not at specific primary sequence sites as with restriction enzymes, it is not possible to detect the junctions of religated noncontiguous fragments simply by inspection. M13 is the vector of choice for dideoxy sequencing for two main reasons. First, M13 bacteriophages are packaged in single strands of DNA, which are extruded from infected Escherichia coli cells into the surrounding culture medium. This means that considerable quantities of single-stranded template DNA can be easily produced. Second, the M13 mp vectors have a quick color assay to identify bacterial cells infected with phage containing an insert.

Correlation of hepatitis B virus DNA and antigens in the liver: A study in chronic liver disease

Hepatitis B virus deoxyribonucleic acid (DNA) and antigens (HBsAg and HBcAg) were studied in liver biopsy specimens from 105 HBsAg-positive patients with chronic liver diseases. Free or integrated viral DNA, or both, was detected in 83 of 105 (79%) patients, whereas HBsAg and HBcAg were demonstrated immunohistologically in 96 (91%) and 39 (37%), respectively. Of 60 patients with detectable free viral DNA, 38 (63%) were positive for HBcAg, whereas only 1 of 45 (2%) with either integrated viral DNA alone (n = 23) or no detectable viral DNA (n = 22) was positive for HBcAg (p < 0.001). Furthermore, the amount of HBcAg was positively correlated with the amount of free viral DNA in the liver tissue. In contrast, HBsAg was well expressed not only in the liver with free viral DNA, but also in the liver with integrated DNA. These data suggest that the synthesis of HBcAg is primarily directed by free viral DNA, whereas that of HBsAg may be directed by free as well as integrated viral DNAs.

Flow Cytometry of Renal Oncocytoma: Common Occurrence of Deoxyribonucleic Acid Polyploidy and Aneuploidy

Flow cytometry was performed on 51 typical specimens of renal oncocytoma. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Of the 51 available tissue blocks 86 per cent were evaluable and 50 per cent of these samples showed a deoxyribonucleic acid (DNA) histogram that was approximately the same as normal renal parenchyma. Of the oncocytoma samples 39 per cent showed a marked increase (more than 10 per cent of the nuclei) in the tetraploid DNA peak, while 11 per cent showed a distinct DNA aneuploid peak. Among 21 evaluable grade 2 oncocytic renal tumors 33 per cent showed a normal DNA histogram, 43 per cent showed a marked increase in the DNA tetraploid peak and 24 per cent showed a DNA aneuploid peak. The common presence of polyploid nuclei containing double quantities of chromosomal DNA may correlate with the long-standing pathological observation that oncocytic tumors often contain a distinct population of large nuclei. Indeed, 86 per cent concurrence was seen between the detection of an abnormal DNA content by flow cytometry and the histopathological presence of large abnormal nuclei in these specimens. Since renal oncocytomas (grade 1 oncocytic tumors) rarely, if ever, metastasize and are relatively noninvasive locally, their markedly abnormal flow cytometry patterns are of considerable interest. Moreover, DNA polyploidy has not been identified previously in renal tumors. The biological significance and mechanism of DNA polyploidy, and the relationship of DNA polyploidy and DNA aneuploidy to the pathogenesis of oncocytic renal tumors require further laboratory investigation. The clinical use of flow cytometry to classify and to predict the behavior of renal tumors will be complicated, since renal oncocytomas commonly have polyploid and aneuploid DNA histograms

Techniques of DNA hybridization detect small numbers of mycobacteria with no cross-hybridization with non-mycobacterial respiratory organisms.

The traditional methods used in identifying mycobacteria, such as acid-fast bacillus stains and culture, are often time-consuming, insensitive, and nonspecific. As part of an ongoing program to improve diagnosis and characterization of mycobacteria, we have found that deoxyribonucleic acid (DNA) hybridization techniques using isotopically labeled, single-stranded, total DNA can be used to detect as little as 10(-4) micrograms of Mycobacterium tuberculosis (MTb) DNA. This amount of DNA represents approximately 2 X 10(4) genomes. We have also shown the MTb DNA is sufficiently different from the DNA of non-mycobacterial microorganisms such that cross-hybridization with MTb DNA does not occur under the hybridization conditions we employed. We speculate that DNA hybridization techniques may allow the rapid, sensitive, and specific identification of mycobacteria.

A histochemical study of the metabolism of rat renal arteries and arterioles.

A histochemical study of the metabolism of rat renal arteries and arterioles. Rat renal arteries and arterioles were examined histochemically to determine their metabolic profiles. Succinate, malate and NAD-isocitrate dehydrogenase, cytochrome oxidase and ubiquinone were assessed to determine aerobic metabolism. Glucose-6-phosphate dehydrogenase and DPN diaphorase were evaluated to determine hexose-monophosphate-shunt activity. Anaerobic metabolism was evaluated via lactate dehydrogenase, and the substrate, glycogen. Gomori's lipase, beta-hydroxybutyrate dehydrogenase and amounts of neutral fat and free fatty acids were assessed as indicators of lipid utilization. Myosin ATPase activity was evaluated as an index of ATP utilization for contraction. Deoxyribonucleic and ribonucleic acids were appraised as indicators of protein synthesis. In general, the oxidative enzymes and myosin ATPase demonstrate considerable activity in renal arteries and arterioles which suggests aerobic metabolism and ATP usage. Renal arteries and arterioles also appear capable of anaerobic metabolism as indicated by strong lactate dehydrogenase reactivity and by the presence of slight to moderate quantities of glycogen, while high levels of glucose-6-phosphate dehydrogenase and moderate amounts of deoxyribonucleic acid suggest a potential for beta-hydroxybutyrate dehydrogenase, minimal lipase activity, and the absence of fatty acids with substantial amounts of neutral fat, indicate limited lipid catabolism.

Influence of disulfide-reducing agents on fractionation of the chromatin complex by endogenous nucleases and deoxyribonuclease I in aging mice.

Age-related alterations in chromatin were evaluated by a fractionation procedure involving limited digestion of liver cell nuclei from young adult and old mice with endogenous nucleases and deoxyribonuclease I. The results suggest that in the chromatin the bulk of the nuclear deoxyribonucleic acid (DNA) may be folded or compacted into domains or loops and associated at a few points with a nuclear protein skeleton structure represented by the final pellet fraction which is resistant to deoxyribonuclease I. Disulfide bonds appear to play a role in the linkage of regions of DNA with this nuclear protein skeleton structure. The amount of DNA that could be released from the pellet fraction by disulfide-reducing agents was significantly greater with old than with young adult mice. In addition, the amount of chromatin material released into the first soluble fraction decreased and that in the second soluble fraction increased with age. Treatment of the nuclei with disulfide-reducing agents did not correct this particular age-related change. Previous results had shown a similar alteration with age of chromatin subjected to digestion by micrococcal nuclease. The combined results suggest the existence of a supranucleosomal alteration in chromatin structure during aging.

Possible Relationship Between Yeast Deoxyribonucleases and Deoxyribonucleic Acid Yield

The deoxyribonucleic acid (DNA) yield and deoxyribonuclease (DNase) activity of several yeasts were correlated. Debaryomyces castellii and Debaryomyces franciscae were found to contain active DNases which carry out DNA hydrolysis, whereas the amounts of DNA as determined by extraction with Sarkosyl buffer (pH 7.8) were found to be small. On the other hand, Candida parapsilosis, Saccharomyces carmosousae, and Lodderomyces elongisporus produced no detectable DNases active at pH 7.8, and their DNA yield was correspondingly high. L. elongisporus was found to possess DNase only at pH 4.0.

Cultivation of Virulent Treponema pallidum in Tissue Culture

In a series of seven experiments, the virulent Nichols strain of Treponema pallidum was shown to attach and replicate on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1Ep) growing in conventional monolayer cultures under an atmosphere of 1.5% oxygen. Five days after inoculation of 10 6 T. pallidum , the number of treponemes had increased to between 8 × 10 6 and 2.59 × 10 7 . The viability of harvested organisms ranged from 86 to 97%. The number of T. pallidum continued to increase, generally reaching a plateau between days 9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 × 10 8 irrespective of the inoculum, which ranged from 10 6 to 10 8 T. pallidum. Concurrent deoxyribonucleic acid assays were performed on the cultures containing T. pallidum to obtain further evidence of replication. Significant increases in treponemal deoxyribonucleic acid were observed when the inocula ranged from 10 6 to 10 7 , with the greatest increases, as might be expected, being in the former group. There was also excellent correlation in the amount of deoxyribonucleic acid per treponeme; the averages for the 10 6 , 2.5 × 10 6 , and 10 7 groups were 3.46 × 10 −14 , 3.28 × 10 −14 , and 2.79 × 10 −14 g per treponeme, respectively (3.14 ± 0.72 × 10 −14 g per treponeme). In each experiment, organisms were harvested from the group inoculated with 10 6 T. pallidum after 7 days of incubation to test for virulence. In all instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms. Scanning electron microscopy revealed that during the course of replication many microcolonies of treponemes formed on the surface of the cells.

Quantitation of bacillus subtilis L-form growth parameters in batch culture.

Several methods were used to monitor the growth of a stable L-form in batch culture. The end of the exponential growth phase was determined with greatest accuracy by the amounts of deoxyribonucleic acid per milliliter of culture. Optical density and viable count data were not as reliable because the L-forms began to lyse at the end of exponential growth. Lysis was detected visually, by phase-contrast observations of wet mounts, and by release of ultraviolet-absorbing material into culture supernatant fluids.

A Study of Kinetoplast DNA in Trypanosoma Equinum

Members of the Protozoan Order Kinetoplastida are characterized by the presence of a unique organelle called the kinetoplast. The kinetoplast, a Feulgen-positive granule, is an integral part of the single mitochondrion of these flagellates and contains a large amount of deoxyribonucleic acid (DNA, Fig. 1). Dyskinetoplasty, an apparent disruption of the kinetoplast DNA (kDNA), occurs spontaneously at low frequency in different species of Kinetoplastida, and can be induced experimentally with various DNA-complexing compounds (1). Ultrastructural studies of the equine pathogen Trypanosoma equinum, a naturally occuring totally dyskinetoplastic species, were undertaken to determine the possible presence, morphology, and location of kDNA within the mitochondrion.

Chromosome replication in sporulating cells of Bacillus subtilis.

A method of specifically labeling the chromosomal terminus of Bacillus subtilis is described. When sporulating cultures were pulse-labeled with [(3)H]thymidine and then treated with 6-(p-hydroxyphenylazo)uracil, a drug which inhibits deoxyribonucleic acid (DNA) synthesis rapidly and completely, the only labeled spores formed were those that had completed replication during the pulse period. DNA-mediated transformation was used to show that the DNA of spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same ratio of origin to terminus markers as DNA from untreated spores. Furthermore, spores formed in the presence of 6-(p-hydroxyphenylazo)uracil had the same DNA content as untreated spores. These two observations indicated that spores formed in the presence of 6-(hydroxyphenylazo)uracil contained completed chromosomes. The rate of termination of chromosomes destined to be packaged into spores was determined by this method, using the Sterlini-Mandelstam replacement system and a single medium exhaustion system for inducing sporulation. With both systems the rate of termination reached a broad peak 2 h after the start of sporogenesis. This was measured from the time of resuspension by using the replacement system and from the point where exponential growth ceased in the exhaustion system. The amount of spore DNA synthesized in the Sterlini-Mandelstam sporulation-inducing medium was very close to one-half the amount of the DNA present in mature spores. This suggests that chromosomes destined to be packaged into spores were replicated from close to the origin and possibly initiated in the sporulation-inducing medium. A method was devised for estimating the time taken to complete replication of the chromosomes destined to be packaged into spores. This was probably no more than 50 min. Whereas starvation must have occurred almost simultaneously in most cells in the population, the chromosome replication that was essential for sporogenesis was distributed over a wide time span. Thus, in some cells, replication started within 10 min of the nutritional step-down, but the peak rate was not reached for 1 h; thereafter replication continued at a substantial rate.

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.

The events in the contralateral lung following pneumonectomy in the rabbit.

Growth of the contralateral lung was studied over a period of 21 days following pneumonectomy in 10-week-old rabbits. Lung volume and lung protein were increased on day 5, and lung weight was increased on day 7 after pneumonectomy. Synthesis of deoxyribonucleic acid (DNA) in the cells of the alveolar wall, as assessed autoradiographically, was first significantly increased on day 5, reached a peak on day 11, and had declined to control level by day 21. Synthesis of DNA also increased significantly in airway epithelial cells between days 5 and 11 with a peak on day 7, but the increase was less marked than in the alveolar wall. The amount of DNA in the lung increased significantly by day 9, and the increment in DNA was about that anticipated from autoradiography. It seems that maximum compensatory lung growth occurs in the second week following pneumonectomy in the rabbit and is complete in 3 weeks. The compensatory growth involves proliferation of both parenchymal and non-parenchymal cells in the alveolar wall.

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.

Deoxyribonucleic acid and outer membrane: strains diploid for the oriC region show elevated levels of deoxyribonucleic acid-binding protein and evidence for specific binding of the oriC region to outer membrane

We have recently reported that part of the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli is associated with the outer membrane fraction and that an outer membrane protein having a molecular weight of 31,000 probably is involved in this association (H. Wolf-Watz and A. Norqvist, J. Bacteriol. 140:43-49, 1979). We have now found that F' merodiploid strains containing two copies of the DNA between bglB and ilv have increased levels of this protein and an increased amount of DNA in their outer membranes. Increased levels of the protein are also found when lambda asn phage, containing at 1.5-megadalton fragment of DNA located to the right of the uncA uncB genes but to the left of oriC, are induced. It therefore seems that this 1.5-megadalton fragment of DNA either codes for or binds to the 31,000-dalton outer membrane protein. Hybridization studies utilizing DNA found to be bound to outer membrane and DNA isolated from a specialized transducing phage lambda asn 132 revealed that at least 5 to 10% of outer membrane DNA has a DNA sequence homologous with a chromosomal segment carried by this oriC-containing phage.

Deoxyribonucleic acid and outer membrane: binding to outer membrane involves a specific protein

The binding of deoxyribonucleic acid (DNA) to the outer membrane of Escherichia coli was examined. The amount of DNA found to be bound to outer membrane was low and was estimated to be about 0.4% of the total DNA. Treatment of cells with chloramphenicol or rifampin caused a disassociation of the apparent DNA-outer membrane complex. The results presented here suggest that the binding between membrane and DNA is specific and involves a membrane protein having a molecular weight of 13,000.

14] Isolation of specific RNAs using DNA covalently linked to diazobenzyloxymethyl cellulose or paper

Publisher Summary This chapter discusses the isolation of specific ribonucleic acids (RNAs) using deoxyribonucleic acid (DNA) covalently linked to diazobenzyloxymethyl (DBM)-cellulose or DBM paper. Substantial amounts of DNA can be linked covalently to the finely divided DBM cellulose or DBM paper. Either form of insoluble DNA can then be used to select specific species of RNA by hybridization. If RNA is radioactive, DNA paper can be used to analyze the fraction of RNA complementary to the insoluble DNA in a procedure very similar to the one originally developed by Gillespie and Spiegelman. Either DNA-cellulose or DNA paper can be used preparatively to select intact, specific RNAs that can be used subsequently for translation in vitro as a template for reverse transcriptase, as a source of pure infectious RNA, or for any other experiment in which pure, intact RNA is required. The chapter describes the purification of intact RNAs and discusses a quantitative analysis of specific labeled RNAs using DNA paper.

Effects of Polychlorinated Biphenyls on Macromolecular Synthesis by a Heterotrophic Marine Bacterium

Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 mug of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.

Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.