Tungsten particle-induced nicking of supercoiled plasmid DNA.

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.

Relaxation, linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles.

The aim of the study was to characterize DNA lesions caused by microprojectile bombardment and by the post-bombardment presence of tungsten particles in transformed cells. For the sake of simplicity, plasmid DNA was used as a target for bombardment with naked tungsten particles. Unexpectedly extensive DNA degradation was observed under standard bombardment conditions. However, no further DNA fragmentation occurred under post-bombardment conditions, simulated by incubation of plasmid DNA with a suspension of tungsten particles. Instead, relaxation and linearization of supercoiled circular plasmids (pAHC25 and others) took place. It is concluded that the observed linearization (a single site double-strand break in DNA circle) results from the ability of tungsten to catalyse the hydrolysis of phosphodiester bonds in torsionally strained sites of native DNA selectively.

Nuclear extrachromosomal DNA of higher plants.

Recent reports indicate that minichromosomes and other small genetic entities may occur in the nuclei of uninfected higher plants. They become especially abundant under some special growth conditions and, sometimes, resemble extrachromosomal genes of ciliated protozoa. An example of such gene-sized DNA species was isolated from resting wheat embryos. The presence of telomeric sequences at its termini and the ability to replicate autonomously in wheat nuclei made it possible to distinguish this nuclear minichromosome from chromosomal DNA fragmentation products. The biological significance of plant minichromosomes remains to be elucidated.

Autonomous replication of a wheat DNA sequence in isolated wheat nuclei.

A fragment of wheat nuclear DNA was shown to be able to replicate autonomously in wheat nuclei. The fragment was 637-bp long and contained telomeric repeats at both termini. Its replication was manifested by the appearance of a radioactive reaction product of the same approximate size. Further analyses showed that the linear, double-stranded reaction product was labelled along its entire length and contained the same number of similarly located HaeIII sites as did the fragment tested. Prokaryotic DNA remained unlabelled under the same assay conditions.

An extrachromosomal fragment of telomeric DNA in wheat.

A procedure developed originally for selective extraction of viral (extrachromosomal) DNA from virus-infected mammalian cells was applied to cell nuclei isolated from uninfected wheat embryos. The resulting nuclear extrachromosomal DNA (exDNA) was enriched for telomere-type sequences by isopycnic centrifugation and inserted into the Sma I site of pUC119. A cloned DNA fragment (241 bp) was found to consist primarily of tandemly repeated heptamere units of the same sequence (5'-CCCTAAA-3') that is known to predominate in telomeric DNA of Arabidopsis thaliana. Hybridization experiments indicate that extrachromosomal telomeric repeats are abundant in resting embryos and disappear rapidly during germination.

A reverse transcriptase-like activity of wheat (Triticum aestivum) embryo microsomal fraction.

A reverse transcriptase-like activity was isolated from germinating wheat (Triticum aestivum) embryos. The activity was found to be associated with a microsomal fraction (70,000 g pellet) of the embryo homogenate. The microsome-associated enzyme prefers homologous polyadenylated RNA to any other polynucleotides as template and requires all four deoxyribonucleoside triphosphates for maximal activity. The reaction product appears in the incubation mixture in the form of an RNA-DNA hybrid, which can be converted into single-stranded DNA by mild alkaline hydrolysis. These observations suggest that normal wheat embryo cells contain an enzyme which, functionally, is similar to retroviral reverse transcriptase.

A mechanism that couples DNA transposition to cell differentiation (hypothesis).

A hypothesis underlying the significance of cytoplasmic events in the process of DNA transposition is proposed. To appear at a new chromosomal site, the transposon sequence is assumed to be first transcribed and then, in a form of the RNA copy, transported to the cytoplasm. A translation-dependent selection of RNA transcripts for reverse transcription is then made and the resulting single-stranded DNA copies return to the nucleus. The free copy may integrate within a new, transcriptionally inactive region of the chromosomal DNA. As a net result, cell differentiation advances in a manner coupled to the message utilization. The hypothesis finds support in many observations, particularly those made for plant cells.

Uptake and binding of cytoplasmic DNA by wheat embryo cell nuclei.

Cytoplasmic non-mitochondrial DNA was selectively labelled and isolated In a linear single-stranded form from early wheat embryos. The isolated preparation was readily taken up by wheat embryo cell nuclei and firmly bound to large, chromosome-like structures. Similarly prepared nuclear DNA fragments, although taken up, remained unbound and underwent a rapid degradation within the cell nuclei. The selective binding of the cytoplasmic DNA indicates that it might be integrated into the nuclear genome.

Isolation of a single-stranded extrachromosomal DNA from germinating wheat embryos.

Newly-synthesized cytoplasmic non-mitochondrial DNA was isolated from wheat embryos which had been germinated in the presence of [14C]-thymidine for a time period not long enough to trigger the first post-dormant round of the nuclear DNA replication. This extrachromosomal DNA fraction consisted of linear single-stranded polydeoxyribonucleotide chains, corresponding in size to approximately 1.1 X 10(6) daltons, and amounted to about 0.5% of the total cellular DNA content. It is suggested that the appearance of the newly-synthesized polydeoxyribonucleotide chains in the cytoplasm may be a physiological signal for the initiation of the nuclear DNA replication in germinating wheat embryo cells.

Oligo(U)- and poly(U)-containing RNA of wheat embryo.

A UMP-rich RNA fraction was separated from the bulk cellular RNA by affinity chromatography of wheat embryo total RNA. The obtained preparation was heterogeneous and contained polyribonucleotide chain segments which were resistant to RNAase T1 and consisted mainly of UMP residues (87 mol%). The UMP-rich segments were of various sizes, including large oligonucleotides and polynucleotides (up to approx. 150 nucleotides in length). The oligo(U)- and poly(U)-containing RNA fraction occurred in a low amount (approx. 1% of total RNA) both in dry and germinating wheat embryos. However, at the onset of germination, labelled precursors were preferentially incorporated into the UMP-rich RNA species. The early-synthesized RNA appeared and underwent a considerable degradation within the cell nuclei. It is assumed that both delayed maturation of structural gene transcripts and rapid transcription of regulatory gene units during initial germination stages contribute to the transient abundance of newly made UMP-rich RNA in the early wheat embryos.

Properties of a terminal deoxyribonucleotidyltransferase isolated from wheat germ.

An enzyme able to catalyse the polymerization of deoxyribonucleotides in a template-independent manner was isolated from dry wheat germ. This activity is associated with a soluble protein which is homogeneous with respect to the molecular weight (approx. 500000) and, under denaturing conditions, dissociates into product of two size classes, 67000 and 45000 daltons respectively. The enzyme-catalysed polymerization can be primed by oligo- as well as poly-deoxyribonucleotides, and is highly efficient (234 nmol/h per mg of finally purified protein) when only one of the four deoxyribonucleoside 5'-triphosphates is present in the incubation mixture. An extension of the 3'-hydroxy termini of polydeoxyribonucleotide chains for approx. 40 nucleotide residues was achieved when non-denatured DNA and [3H]dTTP were used as the primer and substrate respectively. It is concluded that the enzyme isolated from wheat germ shares catalytic properties with the terminal deoxynucleotidyltransferase of mammalian thymus. Unlike that transferase, however, the plant enzyme prefers non-denatured to single-stranded DNA as primer and requires both Mg2+ and Mn2+ ions for maximal activity.

Phosphorylation in vitro and in vivo of the wheat embryo RNA polymerase II.

One of the large subunits (220 000 daltons) of the wheat embryo RNA polymerase II was demonstrated to undergo phosphorylation with [gamma-32P]ATP in a reaction catalysed by a homologous protein kinase preparation. The same subunit was also observed to be phosphorylated in vivo, at the onset of germination. The phosphorylation resulted in a moderate increase of the RNA polymerase activity.

A comparison of the activation of ribosomal RNA synthesis during germination of isolated and non-isolated embryos of Triticum aestivum L.

Incorporation of [(14)C]uridine into ribosomal RNA and ribosomal RNA precursor fractions was studied in isolated wheat embryos and in embryos remaining within the intact grains during germination. In isolated embryos, radioactive 25S and 18S as well as 31S RNA species appeared already within the first 3 h of germination. In non-isolated embryos, radioactivity could be detected in the mature ribosomal and pre-ribosomal RNA fractions only after 6 h and 12 h germination, respectively. It is concluded that transcription of ribosomal sequences is activated almost immediately in isolated wheat embryos but remains repressed for several hours when the embryo germinates under natural physiological conditions.

High molecular weight UMP-rich RNA of germinating wheat embryo.

The synthesis of a relatively homogeneous RNA fraction was observed in germinating wheat embryo. The fraction synthesised within the first 3 h of germination had a high molecular weight (approx. 2.10(6)) and a specific nucleotide composition. In particular, the UMP content was unusually high (47 mol%). The high UMP content resulted probably from the presence of UMP-rich regions within the newly-synthesised polynucleotide chain. Neither ribosomal nor transfer DNA sequences were transcribed at the same time. It is suggested that the newly-synthesised RNA fraction represents an mRNA precursor and is transcribed in the immediate response of wheat embryo to the environmental stimuli to germinate.

Alcoholysis of the endogenous phosphate esters in rats treated with large doses of ethanol.

1. Monoethyl phosphate was isolated from the liver of rats treated with large doses of ethanol. The (14)C- and (32)P-labelled products were obtained when [2-(14)C]ethanol and [(32)P]orthophosphate respectively were used as the radioactive precursors. 2. The isolated ethyl phosphate preparations were identified by their chemical properties, chromatographic behaviour and enzymic hydrolysis, which, for the (14)C-labelled substrate, resulted in a partial recovery of the administered [(14)C]ethanol. 3. The possibility of artifact formation of ethyl phosphate was excluded by suitable control experiments. 4. It is concluded that ethyl phosphate formed in vivo may be a product of phosphate-catalysed alcoholysis of various phosphate esters. The physiological significance of the possible substitution of water by ethanol in reactions catalysed by hydrolytic enzymes under conditions of acute body intoxication with the alcohol is emphasized.