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The mechanism of induction of apoptosis by the novel anti-cancer drug 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigated in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 microM ET-18-OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-18-OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET-18-OCH3 in the presence of the antioxidant alpha-tocopherol. Similar results were achieved when cells were switched acutely to choline-deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosphatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5',6,6'-tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET-18-OCH3 or CD. Taken together, these results suggest that the form of p53-independent apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.

Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 Micron choline (67-73% of cells) than when grown in 70 Micron choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 Micron and 0 Micron choline for 72h. Cells treated with 0 or 5 Micron choline for 72h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24h, cells grown in 0 or 5 Micron choline synthesize DNA more rapidly than those grown in 70 Micron choline. By 72h, the cells grown in 0 or 5 Micron choline ...

Summary Six oligonucleotide probes complementary to the 16S rRNAs of members of the genus Fibrobacter were designed for identification at the levels of genus, species and subspecies. The temperature of dissociation (T d ) of probe-target hybrid was determined for each probe and used to control the stringency of hybridization. Specificity was demonstrated by comparing hybridization to rRNA from target and nontarget species immobilized on nylon membranes. Hybridization to nucleic acid isolated from cattle and goat gut contents suggested greater genetic diversity among this assemblage of fiber-digesting bacteria than now represented in pure culture.

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the α-subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged. Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.

The probability that damage occurs in closely opposed sites on complementary DNA strands increases when DNA is heavily modified with mutagenic agents. Enzymatic excision of the opposite lesions produces DNA double-strand breaks which give rise to genomic rearrangements (deletions, insertions, etc.). Plasmid systems were developed for studying chemical lesions leading to double-strand breaks and the fate of broken plasmid molecules within bacterial cells. Deletions result from the base-pairing of fortuitously located direct repeats flanking the DNA broken ends; as a consequence, the latter are joined, while the DNA fragment between the direct repeats is deleted. Genomic rearrangements arise during the repair of the DNA double-strand breaks, and both events are due to similar repair enzymes which maintain the integrity of the DNA primary structure when conditions are not stressful. A number of genomic rearrangements and point mutations seem to be predetermined by the DNA primary structure.

Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 bp direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec+ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.

Mitochondrial (mt) DNA from eight cytoplasmic male-sterile (cms) lines of sugar beet from different breeding stations was investigated by restriction fragment analysis and Southern hybridization. All cms lines showed similar but not identical restriction and hybridization signal patterns, readily distinguishable from those of fertile (N) cytoplasm. Digestion of the mtDNA with BamHI, EcoRI, SalI, and XhoI revealed distinct differences between the sterile lines, and six subtypes of the S cytoplasm could be distinguished. Differences between the sterile lines were confirmed by hybridization with a \(\overline{\overline {COX}} II\) gene probe revealing minor, line-specific hybridization signals. The data presented provide evidence for the existence of considerable variation within the only commercially used source of cms in the sugar beet, the Owen's type of cytoplasm.

Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned α-subunit of F1-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.

A set of plasmids containing 42, 21 and 31 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 bp direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.

Variations in mitochondrial genome organization and in its expression between fertile, sterile sugar beet lines and fertile nuclear-restored plants were studied. Southern blot hybridization with COXI, COXII, COB and atpA mitochondrial genes as probes showed that changes in the mitochondrial genome organization of sterile lines are associated with variations in the location of COB, atpA and COXII, but not COXI. When the COXII and atpA genes were used as hybridization probes, differences in the primary structure of mitochondrial DNAs from sterile lines of different origin were revealed. Differences in the transcriptional patterns of the three mitochondrial genes (COXI, COXII and atpA) were observed between fertile and sterile sugar beet lines; COB was the only mitochondrial gene whose transcription was identical in both fertile and sterile cytoplasms. The dominant nuclear fertility restorer genes altered the transcriptional patterns of the COB and atpA without affecting those of the COXI and COXII genes; atp A expression was identical in fertile plants and nuclear-restored plants with sterile cytoplasm.

The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 by direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the Kpnl restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung + but not in extracts from ung − E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet + gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung + E. coli strain in comparison with an ung − strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.

The higher-plant mitochondrial (mt) genome differs from the animal genome in many respects. The mt genomes of plants attain 2500 kb, but those of animals do not exceed 18 kb. The plant mt genomes are organised as multiple circular molecules and they contain, in addition to high molecular weight (HMW) DNA, low molecular weight (LMW) DNA, including plasmid-like molecules (Newton, 1988).

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

The study the molecular basis of the origin of complete and mosaic mutants, pBR322 plasmids with damage to one or both DNA strands were constructed by limited chemical modification of plasmid DNA. Damage to one strand of DNA resulted in the induction of predominantly mosaic mutants. Data were obtained indicating that complete mutations arise as a result of damage to both strands in the region of the mutated gene.

The structural organization of sugarbeet chloroplast DNA (ctDNA) was studied by electron microscopic and biochemical methods. As a result, levels of ctDNA packing were demonstrated. It was observed that deoxynucleoproteid (DNP) fibrils released from the chloroplasts after a mild osmotic shock have 13–14 nm nucleosome-like globules on their fibrils. The fibrils form rosettes with loops of a contour length of 15–20 μm. Minicircular plasmid-like DNA molecules with nucleosome-like globules were found in the chloroplasts. By means of monoclonal antibodies against the β-subunit of Escherichia coli RNA polymerase and protein A-colloidal gold complexes, the sites of DNP fibrils to which chloroplast RNA polymerase are attached were localized. The highest number RNA polymerase molecules were found to be associated with the relaxed smooth DNA fibrils which are almost devoid of nucleosome-like granules. The compactly packed DNP fibrils with a high number of nucleosome-like granules were free of RNA polymerase molecules. The former regions of the chloroplast nucleoid are presumably the transcriptionally active sites of the genome, while the latter are the transcriptionally inactive. The RNA polymerase molecules were also found to be attached to the minicircular DNA.

The in vitro binding of 2000-fold purified rat liver glucocorticoid—receptor complexes to functionally differing homologous DNA fractions has been studied. The effectiveness of receptor binding to the transcriptionally active DNA, isolated from rat liver 4 h after cortisol administration, increased by 1.6–1.8-fold with simultaneous reduction in receptor affinity for the hardly extractable DNA fraction. The analogous DNA fractions from control animals did not differ in ability to bind the receptor. This suggests that sites of high affinity for the receptor are directly involved in the in vivo process of hormonal transcription activation.

The kinetics of α-lactalbumin refolding were studied by the stopped-flow method with the registration of CD and intrinsic fluorescence at several wavelengths. It was shown that the early kinetic intermediate forms during the dead-time of the experiment (20 ms). This intermediate has a considerable amount of secondary structure and unpolar clusters in its molecular structure but has no rigid tertiary structure.

Development of a cluster of point mutations due to the correction of an imperfect hairpin in plasmid DNA was investigated. Plasmid pBR322 DNA containing opposite double-strand DNA lesions in the region of a quasipalindrome was constructed. For this aim plasmid DNA was cleaved at the BamHI site, and cytosine residues of the sticky ends were modified by O-methylhydroxylamine. Modified linearized plasmid DNA was ligated and used for transformation of E.coli cells. Tetracycline-sensitive transformants were selected, and the mutants were characterized by restriction and sequencing analysis. One mutant contained a cluster of point mutations. The distribution of mutations was consistent with the cluster having arisen through correction of the imperfect hairpin formed by the quasipalindrome.

It was found that DNA alkylation at the N-7 guanine with the bulky alkylating reagent, N,N,N'-tri-(β-chloroethyl)-N-(p-formylpheny)propylene diamine-1,3 (TFP) is much diminished when DNA is double-stranded. We report here an application of this reaction for probing the hairpin structure in the palindrome-containing single-stranded (ss) DNA fragment of 377 bases prepared from the EcoRI-BamHI fragment of plasmid pBR322. 5'-Labeled ss fragment was modified with TFP and cleaved by piperidine hydrolysis at the alkylated guanine residues according to the Maxam-Gilbert procedure. Guanines in the hairpin formed by palindrome of 9 bp were protected from TFP action, while dimethyl sulfate modified all guanines.

The structure of mitochondrial DNA (mt-DNA) from sugarbeet (Beta vulgaris L.) has been studied by biochemical methods and electron microscopy. It was found to be complex multipartite consisting of two main classes of molecules: high molecules weight (HMW) mtDNA and low molecular weight (LMW) mtDNA. The HMW mtDNA consists of rosette-like structures and globules resembling chromomeres (150-200nm). A typical rosette has a protein core and radially stemming closed DNA loops (from 0.6-1.5 μm). The number of loops in a rosette varies from 16-30. The bulk of HMW mtDNAs are represented by interconnected rosettes (total contour length about 130-160 μm, 403-496 kbp). Such large circular DNAs may be evidence of the master chromosome arrangement of the sugarbeet genome. Globules and rosettes are interconnected by thick and thin DNA fibrils, along which nucleosome- and nucleomere-like structures are distributed. The LWM mtDNA is composed of two groups of supercoiled circular molecules, 0,2-1.5 μm and 0.02-0.05 μm in size. Electrophoretic analysis demonstrated that LWM mtDNA is represented by minicircle plasmid-like DNA molecules of 1.3, 1.4 and 1.6 kbp.

The thymidine derivatives araAzT, dTTP(3′N 3 ), TTP(3′NH 2 ), and araTTP(3′N 3 ), were studied as inhibitors of the reverse transcription taking place within endogenous retroviral A-type particles, where retroviral RNAs served as templates and primers. dTTP(3′N 3 ) was shown to be the most efficient inhibitor of retroviral particle reverse transcription. Termination of DNA chain elongation is the basic mechanism of the inhibitory action of dTTP(3′N 3 ). The compound has a very low inhibitory effect on mammalian DNA-dependent DNA polymerases α, β and γ.