Identification of maize genotypes by PCR analysis]

The possibility of identification and differentiation of maize genotypes by means of RAPD-, SSR- and ISSR-methods was analyzed. DNA of 12 inbred lines and 6 corresponding hybrids of maize were used. Presented methods of the PCR-analysis have allowed unique differentiation and identification of maize genotypes. A formula that describes the results of the DNA polymorphism analysis was proposed.     

Genome Studies by Means of DNA Markers of the Blackcurrant

Russian Journal of Genetics - The paper provides an overview of foreign and Russian studies of the genus Ribes genome by means of DNA markers. A list of methods for DNA extraction from currants is...     

Ultrasound-mediated gene transfer into neuronal cells

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

Grouping of adenoviruses and identification of restriction endonuclease cleavage patterns of adenovirus DNAs using infected cell DNA: simple and practical methods

Simple and practical methods for grouping of adenoviruses and for identification of restriction endonuclease cleavage patterns of viral DNA were established by using infected cell DNA. DNA homology groupings of adenoviruses could be examined by spot hybridization, and restriction endonuclease cleavage patterns of viral DNAs could be obtained by Southern blot hybridization, by using infected cell DNA. The method was very sensitive and allowed the identification of the cleavage pattern of viral DNA of the inoculum by means of cell DNA extracted from infected cells with undetectable cytopathic effect (CPE). In ethidium bromide-stained gels without Southern blot hybridization, the restriction endonuclease cleavage pattern of viral DNA could be detected precisely in spite of background staining due to cellular DNA. The preparation of infected cell DNA used in these procedures was technically much easier than that of viral DNA. These methods require only a small number of infected cells and allow many isolates to be investigated with ease.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

Nuclear disintegration induced by cytotoxic T lymphocytes. Evidence against damage to the nuclear envelope of the target cell

CTL and NK cells induce nuclear disintegration in their target cells. This phenomenon, which is seen as extensive fragmentation and solubilization of target cell DNA, is not seen with most other means of inducing cytolysis, including antibody- and complement-mediated cytolysis. We have previously shown that the degree of DNA solubilization is dependent upon the nature of the target cell. We here investigate the possibility that CTL induce, in all targets, damage to the nuclear envelope, which in turn leads to nuclear disintegration in only some of them. We reasoned that damage to the nuclear envelope would render nuclear DNA more accessible to exogenous DNase. Therefore, we determined the susceptibility of target DNA to exogenous DNase I after cytolysis by various means. We found no difference in DNA susceptibility for cells lysed by CTL vs methods (such as complement-mediated lysis or nonionic detergent) incapable of inducing nuclear disintegration. As a positive control, freezing and thawing dramatically enhanced susceptibility of the DNA. In conclusion, we found no evidence that the nuclear envelope is damaged by CTL in target cell types (or in the subpopulation of nuclei) that do not undergo nuclear disintegration.     

An effective method of DNA isolation from the mature leaves of Gossypium species that contain large amounts of phenolic terpenoids and tannins

Purified and unstained nuclei were isolated from the leaves of several Gossypium species (diploid and tetraploid) by means of a citrate buffer (pH 5.0), Triton X-100 (5%), and a reducing sugar (1M glucose). DNA, previously unobtainable, was then extracted from the nuclei by conventional means. Comparisons of final DNA yield were made between three methods of purification: namely, the standardized ribonuclease procedure, hydroxyapatite chromatography and equilibrium density centrifugation in cesium chloride. The latter method produce the lowest, yet purest, yield of DNA for renaturation studies in Gossypium.     

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.     

Biophysical modeling of radiation induced genetic damage to cells

The paper deals with the new approach for high accurate prediction of ionising radiation induced lesions of the cellular genetic structures. The previous techniques mainly were based on the assumption of the random radiation-induced breakage of the cellular DNA. They did not consider higher-order DNA organisation in the chromatin and in the interphase chromosomes. The paper discusses the new methods of the biophysical modelling of DNA breakage following high LET irradiation which takes into account the information on 3-dimensional structural organisation of DNA in interphase chromosomes. On this basis the influence of DNA organisation in the chromosomes on both dsb clusters induction and on repair were quantitatively studied, that was impossible by the means of previous computational techniques.     

Changes in DNA properties due to treatment with the pesticides malathion and DDVP

Summary Properties of calf thymus DNA were investigated after treatment with the pesticides malathion (0,0-dimethyl-S-(1,2-bis ethoxycarbonyl ethyl)dithiophosphate) and DDVP (0,0-dimethyl-0-(2,2 dichlorovinyl)phosphate) in vitro by means of derivative (differential) pulse polarography (DPP), thermal denaturation curves recorded spectrophotometrically (T_m), viscometric measurements, and chromatography on the hydroxyapatite column. Changes in the properties of DNA were observed by means of DPP after only a few hours incubation with the pesticides, whereas the other methods did not detect any changes even after 48 h. The results obtained by DPP indicate that single-stranded segments and thermolabile regions are formed in DNA due to the action of the pesticides. This behaviour could perhaps be a consequence of guanine alkylation followed by depurination and chain scission at elevated temperatures. Malathion and DDVP differ in the kinetics of reaction with double-helical DNA. DDVP is more reactive and its action is also manifested after 72 h in changes in viscosity, T_m, and chromatographic behaviour on the hydroxyapatite column. The changes induced by malathion were, under identical conditions, not detectable by these methods.     

DNA interaction with low molecular weight ligands of different structure. III. DNA complexes with distactins

The DNA complexes with distactins have been investigated by means of spectrophotometry, viscosimetry and flow birefringence methods. The distactins are actinocin's derivatives containing in the 1,9 positions of the phenoxazone moiety oligopyrrolcarboxamide groups (like those of distamycin A), which have from one to three fragments of 1-methyl-4-amino-2-pyrrolic acid. The mode of DNA-distactins binding in water solution depends on the quantity of the methylpyrrole rings in the oligopeptide groups. The ligand with oligopeptide groups containing three methylpyrrole rings joins the DNA double helix only from outside by means of oligopeptide groups. The compounds with one and two methylpyrrole rings form two kinds of complexes with DNA: external binding and intercalation. In the latter case both chromophore and methylpyrrole fragments, interact with DNA.     

Cytoplasmic Nucleoids in the Generative Cell,Sperm Cell and Egg Cell of Calystegia heder acea

Cytoplasmic nucleoids in the generative cell of mature pollens, sperm cells of pollens cultured in vitro and egg cell of mature embryo sac in Calystegia bederacea Wall. were studied by means of the DNA fluorochrome DAPI in conjunction with epitluorescence microscopy for in situ detection of cytoplasmic DNA in cells. Results showed that many cytoplasmic DNA nucleoids were present in the generative cell and speim cells. Two types of nucleoids were observed, one with big and strong fluorescent dots, and the other with small and weak fluorescence. Many dot-shaped and a few circle-shaped nucleoids were randomly distributed in the thin layered cytoplasm of the egg cell. It was suggested that different types of nucleoids might represent plastid DNA and mitochondrion DNA respectively. Results provided cytological data that Calystegia hederaeea had the potential of plastid DNA biparental inheritance, and the mode of which merits further study via molecular biological methods.     

打碗花生殖细胞,精细胞及卵细胞中的细胞质类核

已有不少超微结构的资料阐明被子植物双亲和单亲母系质体遗传的细胞学基础。近年应用DAPI荧光染色的方法,可快速地从检测质体DNA存在的状况确定被子植物中具双亲遗传潜能的种。从质体的类核存在与否判断质体遗传方式为母系遗传或双亲遗传与已有的遗传分析结论基本一致,只有少数种类是矛盾的。DAPI荧光技术可以认为是研究细胞质遗传机理的一个重要手段。我们曾证明旋花科牵牛属植物生殖细胞、精细胞中存在细胞质类核,确定其具双亲或单亲父系质体遗传的潜能,并用RFLP技术进一步确定其为质体父系遗传型。本研究证明旋花科的打碗花属生殖细胞、精细胞和卵细胞中细胞质类核存在的状况与牵牛属的相似,提供了打碗花可能在质体遗传上与牵牛属 具相同的遗传方式的资料。     

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.     

Towards more efficient large-scale DNA-based detection of terrestrial mammal predators from scats

The DNA detection of wildlife from environmental samples has the potential to contribute significantly to wildlife management and ecological research. In terrestrial ecosystems, much work has focused on the identification of mammal predators from faecal (scat) samples. However, the relatively high time and financial costs of collecting and analysing scat DNA remain barriers to more widespread implementation of such DNA detection methods, especially for high-throughput surveys. Here, we evaluate methods used for DNA extraction from scats, as applied to detection of the Australian red fox, an introduced predator. We compare the relative costs of two approaches: the method previously used to screen thousands of scat samples in surveys over several years, and a modified version which involves swabbing scats at the time of collection and using a mechanised liquid handling platform to extract DNA from the swabs. We demonstrate that mechanised DNA extraction from swabs is more efficient than manual DNA extraction from whole scats, in terms of both time and resources. This provides a means for rapid, high-throughput screening of scats for the presence of mammal predators, enabling time-effective management responses to non-invasive surveys.     

Structure of DNA complexes with regular polypeptides

The conformation of some regular polypeptides: (Lys-Ala)50, (Lys-Ala2)37, (Lys-Ala2)26, (Lys-Ala3)18, (Lys3-Pro)29, (Orn3-Gly)28 was studied by means of CD. The complexes of these polypeptides with DNA were obtained by the methods of jump-dilution of a two-components mixture from 2 M NaCl to 0.05 M NaCl. The extent of DNA covering by the polypeptides was compared using binding isoterms of ethidium on DNA and DNA-polypeptide complex. The length, L, which polypeptides cover on DNA was estimated by means of energy transfer between the dyes absorbed on the complexes. The CD spectra of the complexes revealed a high sensitivity to changes of the environmental conditions. Small variations in the temperature and ionic strength produces marked changes in the CD spectra of the complexes. It was suggested that observed CD changes are due to both the structural relaxation of the complexes and the existence of liquid-crystal domains in solution.