Filtration-fluorescence determination of DNA sedimentation profiles

A method for determination of DNA sedimentation profiles in density gradients of sucrose or salts is proposed. The method consists in isolation and purification of DNA from the fractions by molecular filtration and a subsequent determination of DNA content by the fluorescence of the DNA-ethidium bromide complex.     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: DNA diversification in the evolution of four orchids

The species- and genus-specific DNA content, average base composition of nuclear DNA, presence or absence of satellite DNA, the percentage of heterochromatin and other characteristics of nuclear DNA and nuclear structure allow to deduce the molecular changes which accompanied, or more probably caused, cladogenesis in the orchids studied. It is suggested that saltatory replication (generative amplification) of certain DNA sequenes, diversification of reiterated DNA sequences, and loss of DNA play an important role in the evolution of orchids.—The relationship between changes of genome composition and of nuclear structure and ultrastructure is discussed on the basis of cot curves, heterochromatin staining with Giemsa (C banding), electron microscopy of nuclei, and molecular hybridization in situ.Some aspects of this paper have been presented at the Helsinki Chromosome Conference, August 1977 (Nagl & Capesius 1977).     

DNA contents of replication without DNA density labeling

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.     

Estimation of residual DNA in Feulgen reaction. A new correction of DNA determination per nucleus.

For the determination of the residual DNA amount after acid hydrolysis of Feulgen's method, a high salt-fluorochrome assay for DNA (5 microM Hoechst 33258 with 1 M NaCl) was effectively applied. At an optimal time length of acid hydrolysis for Feulgen reaction, the ratio of the residual DNA of non-hydrolysis to total DNA is 10% or more in hepatocyte or lymphocyte nuclei. A lot of residual DNA seems not to be negligible in Feulgen's method. A more accurate determination of DNA can be made by correcting the loss ratio of the residual DNA value to Feulgen DNA value. Thus, the combination assay of Feulgen's method with the present fluorometry is enough to measure separately both the amounts of Feulgen DNA and its residual DNA and successfully determines more accurately the total DNA per nucleus by summing both the amounts. The residual DNA, a resistant portion of the chromatin DNA against acid hydrolysis, is a possible constituent as the physiological component of nuclear structures.     

Linear kalilo DNA is a Neurospora mitochondrial plasmid that integrates into the mitochondrial DNA

Summary The linear autonomous form of kalilo DNA (previously called AR-kalDNA) is shown to be resident within mitochondria rather than nuclei, as had been suggested by previous experiments. This form has been renamed mtAR-kalDNA, to signify its mitochondrial location. Experiments are described that illustrate the inheritance and somatic transmission patterns of the mitochondrial kalilo plasmid and the mitochondrial inserted form of kalilo DNA (mtlS-kalDNA). Progeny of a cross with a pre-senescent subculture as the female parent inherited mtAR-ka1DNA only; mtIS-kalDNA was not transmitted sexually. During somatic propagation of the ascospore cultures, novel kalilo DNA inserts appeared and most of them persisted until death. We propose that these inserts originated from de novo integration of mtAR-kalDNA into the mitochondrial DNA. In two of the ascopore-derived series analyzed, the first inserts detected were seen only transiently and inserts appearing subsequent to the transient inserts were retained until death. We propose that these enduring inserts originated either from rearrangements of the transient inserts or from novel integration events, either from mtAR-kalDNA or from transposition of the transient inserts.     

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

DNA methylation increases throughout Arabidopsis development

We used amplified fragment length polymorphisms (AFLP) to analyze the stability of DNA methylation throughout Arabidopsis development. AFLP can detect genome-wide changes in cytosine methylation produced by DNA demethylation agents, such as 5-azacytidine, or specific mutations at the DDM1 locus. In both cases, cytosine demethylation is associated with a general increase in the presence of amplified fragments. Using this approach, we followed DNA methylation at methylation sensitive restriction sites throughout Arabidopsis development. The results show a progressive DNA methylation trend from cotyledons to vegetative organs to reproductive organs.     

Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Chromosomal DNA variation in Cucumis

Summary Variation in nuclear DNA amounts found in different species of Cucumis was surveyed. The DNA amounts varied from 1.373 to 2.483 pg in diploids and from 2.846 to 3.886 pg in tetraploids. DNA amount was not correlated with chromosome number and periodicity. Tetraploids were found to have double the quantity of nuclear DNA of diploids. A positive linear relationship was established between the nuclear DNA amounts and volume of chromosomes. The botanical varieties within a particular species do not differ significantly for 2C DNA amounts. A comparison of the distribution of DNA amounts among different chromosomes of haploid complement in different species revealed that the quantitative DNA changes associated with speciation affected all chromosomes. DNA changes were not however, of the same magnitude in all chromosomes of the complement. Speciation in Cucumis thus seems to have occurred through amplification or diminution of DNA proportionate to the size of chromosomes. The relationship between the basic numbers, x=7 and x=12, will have to be considered relative to the high DNA amount noticed in some species with x=12.     

An association between chloroplast DNA haplotypeand gender in a plant metapopulation

Theoretical models have shown that metapopulation structure can influence the evolution of the sex ratio in gynodioecious plants when the fitness of females and/or hermaphrodites varies among populations with the local sex ratio. Such frequency-dependent fitness has been demonstrated previously in natural populations of Silene vulgaris, a gynodioecious plant in which sex determination is cytonuclear. We investigated whether there was an association between chloroplast DNA (cpDNA) haplotype and sex expression, with the assumption that cpDNA haplotypes could be associated with specific male sterility variants likely found in the mitochondrial DNA, owing to co-transmission of the two cytoplasmic genomes. Two cpDNA haplotypes were studied in a collection of 111 individuals of known sex taken from 20 natural populations found in Virginia, USA. The set of haplotype 1 individuals consisted of 71.4% hermaphrodites, where as only 7.7% of the haplotype 2 individuals were hermaphroditic; a highly statistically significant difference. By extension of a previous model it was also shown that the differential clustering of these two haplotypes into local populations contributes to among-population sex ratio variation and has the potential to lower the fitness of haplotype 2 by greater than 50%, relative to that expected with no population structure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

DNA adduction and mutagenic properties of acrylamide

This review article summarizes our current knowledge on DNA damaging and mutagenic properties of acrylamide. Direct and indirect modes of interaction of acrylamide with DNA are discussed, and the resulting alkylating DNA adducts are highlighted. Emphasis is placed on glycidamide-DNA adducts generated via epoxidation of acrylamide presumably by cytochrome P4502E1. Dosimetry and mapping of acrylamide-induced DNA adducts in vitro and/or in vivo are described. Mutagenic potency and specificity of acrylamide in relation to its respective DNA adducts are discussed. Prospective views are provided on the potential applications of acrylamide-induced DNA adduct dosimetry/mapping and mutation frequency/spectrometry for biomonitoring purposes.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Gapped DNA and cyclization of short DNA fragments

We use the cyclization of small DNA molecules, approximately 200 bp in length, to study conformational properties of DNA fragments with single-stranded gaps. The approach is extremely sensitive to DNA conformational properties and, being complemented by computations, allows a very accurate determination of the fragment's conformational parameters. Sequence-specific nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nt in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity. This makes it very attractive to use gapped DNA in the cyclization experiments to determine DNA conformational properties, since the gap eliminates oscillations of the cyclization efficiency with the DNA length. As a result, the number of measurements is greatly reduced in the approach, and the analysis of the data is greatly simplified. We have verified our approach on DNA fragments containing well-characterized intrinsic bends caused by A-tracts. The obtained experimental results and theoretical analysis demonstrate that gapped-DNA cyclization is an exceedingly sensitive and accurate approach for the determination of DNA bending.     

Determination of DNA base composition by small scale acrylamide-CsCl gradient centrifugation

In this paper we describe a simple and rapid protocol for DNA base composition determination by CsCl gradient in the presence of acrylamide. This method permits the determination of GC content in microgram amounts of DNA, and results are easily documented in photographs or graphs. The protocol was applied to the characterization of nematode DNA, but can be used for other organisms. Analyzing several experiments the mean standard deviation observed in the calculated GC content is near 1.3%.     

Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates

Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg²⁺ cations in the formation and stabilization of the microsphere structure was established.