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1.
Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation 总被引:18,自引:0,他引:18
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Walter F. Bodmer 《The Journal of general physiology》1966,49(6):233-258
Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 106. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (2H15N 3H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis. 相似文献
2.
A. R. Lehmann 《Biophysical journal》1972,12(10):1316-1325
Alkaline sucrose sedimentation studies of DNA from mouse lymphoma cells (L5178Y) treated with caffeine have demonstrated the following effects. Caffeine (at a concentration of 1.6 mM) does not introduce strand breaks into preformed DNA nor does it inhibit the rejoining of γ-ray-induced strand breaks. Although it does not affect the over-all rate of DNA synthesis, pulse labeling experiments show that the DNA strands synthesized in its presence are smaller than those made in its absence. This could be the result of (a) DNA being made in shorter replicating units or (b) small gaps in the daughter DNA strands within normal-sized replicating units. These two alternative models were indirectly distinguished as follows. After a pulse label with thymidine-3H in the presence of caffeine, cells were incubated without caffeine in bromodeoxyuridine (BrdUrd). During this incubation, growing strands are elongated and hypothetical gaps (model b) filled in with bromuracil (BrUra)-substituted DNA. The BrUra-containing DNA segments will now be of different lengths on the two models. With smaller replicating units (a) the “elongation segments” will be somewhat smaller than but the same order of magnitude as those in untreated cells, whereas with small gaps (b) the “filled-in gap segments” would be expected to be at least an order of magnitude smaller. The BrUra-containing regions of DNA can be selectively broken open by exposing the cells to light at 313 nm. The exposure required to break open the BUra-substituted regions is inversely related to, and hence gives a measure of, the size of these regions. In caffeine-treated cells these regions were found to be somewhat smaller than but of comparable size with those in untreated cells; this is consistent with the DNA being synthesized in smaller units and argues against the presence of small gaps in the daughter strands. 相似文献
3.
Segregation of Deoxyribonucleic Acid in Bacteria: Association of the Segregating Unit with the Cell Envelope 总被引:10,自引:7,他引:3
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Cells of the gram-positive organism Lactobacillus acidophilus R-26 were labeled with 3H-thymine to measure the segregation of radioactive deoxyribonucleic acid (DNA) into daugher cells. Such cells were found to contain 8 conserved units of DNA which would correspond to two replicating chromosomes per cell. Fluorescent antibody (FA) against this organism was used to demonstrate that portions of the cell surface (2 to 4 units per cell) were conserved during growth and division. The permanent association of DNA with these conserved cell surface units was measured by combining autoradiography with FA techniques. DNA synthesized immediately before FA labeling was not associated with the fluorescent cell surface, whereas DNA synthesized a generation previously was. The results are consistent with a model in which DNA becomes permanently fixed to the cell surface when it is first used as a template. 相似文献
4.
Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli. 总被引:8,自引:0,他引:8
The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from 15NH4+, D2O to 14NH4+, H2O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is , where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass. 相似文献
5.
HeLa nuclear DNA sediments as a single peak, in neutral CsCl, while it is separated in a heavier and a lighter components, in alkaline Ag+–Cs2SO4. The heavy fraction, on the average, represents about 20% of the total DNA. CsCl analytical ultracentrifugation shows that heavy DNA bands at 1.715 g/cm3 and contains 53% GC (10% of the total GC), whereas light DNA bands at 1.703 g/cm3 and contains 40% GC (32% of the total GC). Coherently, Tm values in 0.1 x SSC are 82.5°C, for heavy DNA, and 72.5°C, for light DNA. After treatment with [3H-methyl-S-adenosyl-L-methionine in isolated nuclei, the concentration of labelled 5-methylcytosine was found to be highest in the more dense regions of the heavy peak and in the less dense regions of the light peak. Exposure to ultrasound modifies the quantitative relationship of the two peaks and improves the separation of supermethylated AT- and GC-rich DNAs. Four possible triplets as sites for DNA-methylase recognition are discussed. 相似文献
6.
To evaluate the effects of prolonged moderate body deuteration on incorporation of tritiated thymidine (3HTdR) into the DNA of major cell renewal systems, young adult mice were given drinking fluid containing 30 % heavy water for 7, 14, 21, 42 and 70 days. Control mice drank tap water. Three hours prior to sacrifice, 925 kBq of 3HTdR were injected intravenously. Following extraction of the bulk of the soluble activity with an aqueous formalin solution, the residual 3H-activity of the organs was assayed by liquid scintillation counting and by autoradiography. The total thymic 3H-activity and the thymic weight, particularly of the cortex, were significantly reduced in deuterated mice early in the course of the experiment. The fraction of labeled thymocytes diminished to less than one half of the control values of day 70. The 3H-activity of the bone marrow in deuterated mice was reduced to about of control values. In contrast, the total 3H-activity of the small intestine, as well as mean labeling index and mitotic index of small intestinal epithelia, were not markedly altered in deuterated versus control mice. Drinking water containing 30 % of heavy water did thus not result in generalized, profound and progressive disturbance of 3HTdR incorporation in all the major cell renewal systems in the mouse. The thymus and, to a lesser extent, the bone marrow, were unquestionably affected, but the depression of 3HTdR incorporation did not increase markedly in the course of 10 weeks. In these terms, the toxicity of 30 % heavy water in the drinking fluid appears minor. This is of particular interest since exposure to similar concentrations is known to elicit immunodepressive and/or antineoplastic effects. 相似文献
7.
The marine diatom Amphora sp. was grown at a light intensity of 7.0 × 1015 quanta centimeter−2 second−1. Light saturation of photosynthesis for these cells was between 6.0 and 7.0 × 1016 quanta centimeter−2 second−1. At light intensities greater than saturation, photosynthetic 14CO2 fixation was depressed, while P700 unit size (chlorophyll a concentration/P700 activity) increased and number of P700 units per cell decreased. After a 1-hour exposure of Amphora sp. to a photoinhibitory light intensity of 2.45 × 1017 quanta centimeter−2 second−1, there was a 45 to 50% decrease in the rate of 14CO2 fixation relative to the rate at the culture light intensity. There also was a 25% increase in P700 unit size and a 30% reduction in the number of P700 units per cell but no change in total chlorophyll a concentration. Following this period of photoinhibition, the cells were returned to a light regime similar to that in the original culture conditions. Within 1 hour, both number of P700 units per cell and P700 unit size returned to levels similar to those of cells which were kept at the culture light intensity. The rates of photosynthesis did not recover as rapidly, requiring 2 to 3 hours to return to the rate for the nonphotoinhibited cells. Our results indicate that a decrease in P700 activity (with a resultant increase in P700 unit size) may be partially responsible for the photoinhibition of algal photosynthetic carbon dioxide fixation. 相似文献
8.
Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil 总被引:5,自引:2,他引:3
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David R. Singleton Sabrina N. Powell Ramiah Sangaiah Avram Gold Louise M. Ball Michael D. Aitken 《Applied microbiology》2005,71(3):1202-1209
[13C6]salicylate, [U-13C]naphthalene, and [U-13C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [13C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the 13C during the incubation did not appear to be an issue in this experiment. 相似文献
9.
The role of the infecting viral strand in the replication of bacteriophage φX174 replicative form DNA was studied by [3H]thymidine pulse-labeling Escherichia coli cells infected with 2H15N density-labeled phage. The products of a round of semi-conservative replicative form replication (in light medium) do not contain the original heavy viral strand by 15 minutes after infection or later in the presence of chloramphenicol. Similar results were obtained at earlier times in the absence of chloramphenicol. We conclude that the parental viral strand need not be conserved in the replicating DNA structure in succeeding rounds of replication. 相似文献
10.
Transformation of Leuconostoc carnosum 4010 and Evidence for Natural Competence of the Organism
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Sren Helmark Mette E. Hansen Birthe Jelle Kim I. Srensen Peter R. Jensen 《Applied microbiology》2004,70(6):3695-3699
Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 × 105 transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 × 10−6 to 19 × 10−6 transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent. 相似文献
11.
Experiments were conducted to examine the development of photosynthetic carbon metabolism in Peperomia scandens, a tropical epiphyte. Leaves were sampled during a 10-day period when they were between 30 to 165 days old. P. scandens exhibits a C3 to CAM-cycling to CAM shift during maturation with the magnitude of CAM increasing with age. Initially, during both day and night, no significant CO2 uptake or diurnal acid flux was evident. C3 gas exchange was detected at 41 days of age with a gradual shift towards CAM gas exchange maximized thereafter. An acidity flux of 130 to 150 microequivalents per gram fresh weight was evident by 41 days. Between 40 and 90 days, the leaves shifted their CO2 uptake pattern from a daytime to a nighttime peak. After 90 days, the leaves remained in CAM. The δ13C values became progressively less negative as the leaves matured. In the 30-day-old leaves, the δ13C value was −21.1% while in the 165-day-old leaves the δ13C value was −18.3%. The time-dependent shift from C3 to CAM-cycling to CAM in P. scandens does not appear to result from changes in water, light, or temperature regimes since these variables were constant for all leaves sampled. 相似文献
12.
Cycloheximide causes inhibition of semiconservative DNA replication in HeLa cells by reducing the average rate of DNA chain elongation. 2,4-Dinitrophenol inhibits semiconservative DNA replication (50 to 80% inhibitions at 10?3 to 5 × 10?3 M-2,4-dinitrophenol) without affecting the average rate of DNA chain elongation. Therefore, at any given time the number of replicating sections of DNA per DNA-synthesizing (S-phase) cell appears to be reduced in the presence of 2,4-dinitrophenol.Radioactivity profiles of pulse-labeled DNA in alkaline sucrose gradients suggest that 2,4-dinitrophenol modifies initiation and termination patterns of replicating sections, most of which are found to be 10 to 80 μm (mode: 15 to 30 μm) under control conditions. DNA synthesized in the presence of 2,4-dinitrophenol has the density of control DNA, is metabolically stable, and after mitosis, functions normally as a template in the next round of replication. 相似文献
13.
Specimens of Biomphalaria glabrata, NIH albino strain, challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 109 bacteria/ml, showed significant elevation of serum aminopeptidase activity level at 2 hr postchallenge when compared with the serum activity levels of the enzyme in snails of the other experimental groups at four time intervals postchallenge. It was found that challenge with a heavy dosage of heat-killed bacteria did not cause a shift in the highest level of enzyme activity from 2 hr postchallenge. Also, the activity of aminopeptidase was higher at 2 hr postchallenge with a greater dosage of heat-killed bacteria. 相似文献
14.
Ecological Consequences of Long-Term Exposure of Anabaena variabilis (Cyanophyceae) to Shifts in Environmental Factors 总被引:2,自引:0,他引:2
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Cultures of Anabaena variabilis were exposed to different light intensities, and the time course of photoadaptation was measured by photosynthetic rate and changes in pigmentation. A shift down in intensity of 284 μEin · m−2 · sec−1 caused a temporary decrease in the photosynthetic response followed by gradual adaptation to the new conditions. Final chlorophyll a and carotenoid concentrations were reached after 1 day, although other physiological indicators showed that adaptation required 4 days. The parameter Ik was shown to be the best indicator of photoadaptation. A shift up in light intensity of the same magnitude also required 4 days for complete photoadaptation by the culture, although chlorophyll and carotenoid concentrations stabilized within 1 day. A shift down in light intensity of 392 μEin · m−2 · sec−1 resulted in a temporary attempt to adapt followed by collapse of the population. This demonstrates an apparent threshold in the magnitude of the shift in light intensity which will permit successful adaptation. Simultaneous changes in light intensity and temperature also adversely affected culture populations. Our observations present a possible cause for the decline or prevention of an algal bloom under a fluctuating light regime and suggest that it may be possible to predict this decline as a result of synoptic weather patterns or hydrodynamic influences. 相似文献
15.
Morten E. Allentoft Matthew Collins David Harker James Haile Charlotte L. Oskam Marie L. Hale Paula F. Campos Jose A. Samaniego M. Thomas P. Gilbert Eske Willerslev Guojie Zhang R. Paul Scofield Richard N. Holdaway Michael Bunce 《Proceedings. Biological sciences / The Royal Society》2012,279(1748):4724-4733
Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10–6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R2 = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone. 相似文献
16.
Manuel Snchez Francisco J. Iglesias Carlos Santamaría Angel Domínguez 《Applied microbiology》1993,59(7):2087-2092
The physical and biological parameters involved in efficient transformation of Kluyveromyces lactis by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 106 and 107 transformants per μg of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid. 相似文献
17.
Unsheard chromatin isolated from sea urchin embryos was submitted to buoyant density centrifugation in sucrose-glucose gradients. The main peak of blastula chromatin was at a density position of 1.299±0.028±0.009 g ml-1 whereas at gastrula stage a shift to a lower buoyant density position of (1.276±0.021±0.007 g ml-1) was observed. Besides the main peak, a small band with a density of 1.18 g ml-1 was noticed. The lighter fraction differed from the heavy one in a higher histone to DNA ratio, a lower proportion of the F-1 histone, and a lower nonhistone to DNA ratio. The most pronounced developmental alterations of proteins were observed at the level of nonhistone protein patterns of the light fractions. 相似文献
18.
Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with 3H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of 3H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of 3H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous 3H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of 3H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of 3H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis. 相似文献
19.
The fate of 3H-thymidine-labeled adenovirus type 12 deoxyribonucleic acid (DNA) was studied in Nil-2 cells of Syrian hamster origin. It was found that a substantial fraction of 3H-adenovirus type 12 DNA became degraded within 24 hr after infection and was released into the culture fluid. After infection of 5-bromodeoxyuridine (BUdR)-prelabeled cells with 3H-adenovirus type 12, viral DNA became readily separable from cellular DNA by equilibrium centrifugation in CsCl. Part of the viral radioactivity was found to shift gradually to the position of cellular DNA as time progressed after infection. When exponentially growing cells were exposed simultaneously to BUdR, 5-fluorodeoxyuridine, and 3H-adenovirus type 12, up to 50% of the viral radioactivity shifted within 24 hr from the density of viral DNA to that of cellular DNA after equilibrium centrifugation in CsCl. Upon denaturation of the cellular DNA, the isotope was preferentially found to be associated with the “heavy” strand which was synthesized after infection. Upon hybridization of the “heavy” and the “light” strands with sonically treated, denatured 3H-adenovirus type 12 DNA, small and nearly equal amounts of counts hybridized with both strands. The number of counts annealed was in a range similar to that of those annealed with the same amount of DNA derived from adenovirus type 12-transformed hamster cells. These results demonstrate that (i) a substantial proportion of the adsorbed virus becomes degraded within 24 hr; (ii) part of the degradation products is reutilized for cellular DNA synthesis; (iii) only a small fraction, mainly fragments, of viral DNA becomes integrated into both the newly synthesized and the parental strands of cellular DNA. 相似文献
20.
Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ3 particle volume The mean volume of rat liver mitochondria was 0.42 µ3 or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 103, and per gram of rat liver was about 11 x 1010. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ3 and from rat kidney cortex, 0.23 µ3. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a3), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement 相似文献