Jerzy Buchowicz

The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination

Abstract The syntheses of main macromolecular substances, in a whole wheat grain allowed to germinate, are triggered in the following order: RNA, protein, DNA. The RNA synthesis, as judged by [2- 14 C]uridine incorporation, is initiated almost immediately after the seeds are exposed to the optimal germination conditions, whereas [1- 14 C]leucine and [2- 14 C]thymidine incorporation begins to occur only 3 and 4 hr later, respectively. The initiation of protein synthesis is accompanied by an apparent cessation of uridine incorporation.

Activity of enzymes involved in pyrimidine metabolism in the germinating wheat grains

Abstract Activities of carbamoyl phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, dihydro-pyrimidinase, uracil phosphoribosyltransferase, uridine phosphorylase, uridine kinase and acid phosphatase were measured in extracts from wheat grains germinated for periods ranging from 0 to 48 hr. With the exception of uridine phosphorylase which appeared only 24 hr after initiation of germination, all these activities were detected in the dry grains. Activities concerned with the synthesis of pyrimidine ring de novo dropped at the onset of germination (0·5–2 hr) and increased thereafter gradually. Activities involved in the degradative pathway were maintained at a roughly constant level during the first 24 hr and rose considerably later on. The results are discussed in relation to the sequence of initiation of RNA and protein synthesis during seed germination.

Rna synthesis during the germination of wheat seed

Abstract Incorporation of [14C]uridine into various RNA fractions of germinating wheat embryo was studied. During the first 3 hr of germination the precursor was incorporated predominantly into a specific component of the RNA (messenger RNA). Neither ribosomal nor transfer RNA were labeled at this time. It is concluded that biosynthetic processes are resumed after the breaking of dormancy in a sequential manner. This sequence begins with the initiation of messenger RNA synthesis.

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention.

An extrachromosomal fragment of telomeric DNA in wheat

A procedure developed orginally 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.

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 postbombardment 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 postbombardment 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.

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.

Purine and pyrimidine bases and nucleosides of germinating Triticum aestivum seeds

Abstract Adenine, guanine, uracil and the corresponding nucleosides were identified in mature wheat grain. On germination, contents of these compounds changed in a regular pattern. Amounts of the free bases rose immediately and remained at the increased levels for a short period (3–6 hr). Nucleoside content decreased at the initial phase and increased sharply 12 to 48 hr after germination.

Characterization of the early synthesized DNA in germinating Triticum aestivum embryos

Abstract A radioactive DNA preparation was isolated from the post-mitochondrial supernatant fraction of thymidine-[ 14 C] fed wheat embryos. The isolated s DNA preparation was similar to cytoplasmic non-mitochondrial DNA of other eukaryotic cells. The buoyant density and frequency of pyrimidine nucleotide clusters found for the s DNA were, essentially, the same as those found for the nuclear DNA. In contrast to DNA that can be leaked from nuclei or other DNA-containing organelles, the s DNA is firmly bound to a protein component. At an early germination stage (6–12 hr), the s DNA is the only newly-synthesized DNA fraction that can be isolated from the embryo homogenate. Considerable synthesis of nuclear and organellar DNA starts 18 hr after the beginning of germination, just prior to the first maximum of the cell divisions. It is concluded that wheat embryo cells contain cytoplasmic non-mitochondrial DNA and are able to resume its synthesis at an early germination stage, prior to the first post-dormant round of nuclear DNA replication.

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

Incorporation of [14C]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.