Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Examination of newly synthesized DNA in Escherichia coli.

Summary The short (0-20S) Okazaki fragments seen upon pulse-labeling E. coli (thy ⁺, deo ⁺) with ³H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut ⁺, thyl ⁺, polA ⁺), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.     

Effect of a newly synthesized compound on delaying pre-harvest sprouting in winter wheat (Triticum aestivum L.)

Plant Growth Regulation - Pre-harvest sprouting (PHS) of wheat commonly occurs in rainy or humid weather conditions before harvest. It not only affects the yield but also decreases the wheat...     

Morphological responses to simulated wind in the genus Brassica (Brassicaceae): allopolyploids and their parental species

Thigmomorphogenesis refers to the widespread ability of sessile organisms to modify their morphology in response to a variety of mechanical stimulations, from direct contact with the stem by insects or other plants to flexure caused by wind, water, or snow. In this paper we investigated the differences in the reaction norms to wind exposure of seven species of the Brassicaceae that constitute a well-studied complex of known phylogenetic relationships. The goals included the characterization of differences between allopolyploids and their parental species and the comparison of wild and fast-cycling accessions within each species. We found statistically significant variation for plasticity among species or accessions for several characters, but the majority of the phenotypic variance was accounted for by overall (across-environment) differences among species and accessions and not by variation in plasticity. Allopolyploids displayed an array of behaviors when compared to their parents, from co-dominance to complete dominance to exceeding both parental means. Furthermore, fast-cycling plants showed distinct features from their wild relatives, suggesting that wild populations should be included with artificially selected lines in ecological studies. We proposed further steps to gain a more comprehensive understanding of thigmomorphogenetic responses, by integrating current research on the molecular bases of thigmomorphogenesis with insights into the ecology and evolution of plants exposed to wind.     

Identification of a repeat sequence of rye DNA in wheat and related species

Polymerase chain reaction (PCR) and enhanced chemiluminescence (ECL) were used to determine the distribution of the rye-specific sequence contained in the pSc119.1 probe among wheat and related species. A specific pair of primers targeting this rye-specific sequence was used. A 745-bp fragment, the predicted size of pSc119.1, was present inSecale cereale, Triticum aestivum, XTriticosecale, Hordeum vulgare, H. bogdanii, andH. parodii. PCR results were verified by hybridizing the rye-specific probe pSc119.1 to dotblots of DNA from the different species used. Strong hybridization signals detected by ECL were consistent with the PCR results. The results demonstrate the effectiveness of PCR and dot-blot ECL in screening plants for defined DNA sequences, and indicate that pSc119.1 has counterparts with strong homology inT. aestivum, H. vulgare, H. bogdanii, andH. parodii.     

Methylation of newly synthesized DNA in mouse fibroblast culture]

After a 10 min- or more prolonged incubation of transformed mouse fibroblasts (L.-cells) with [3H]-thymidine or [3H-methyl]-methionine and a subsequent centrifugation of cell lysates in an alkaline sucrose gradient the DNA radioactivity is detected in long (28, 33 and 45S) and short (5, 13 and 18S) fragments. An increase in cell concentration in the cultural layer results in inhibition of 5S fragments linkage rather than in inhibition of their synthesis. The blocking of the Okazaki fragment linkage may be regarded as one of the inhibitory molecular mechanisms of cell depletion. Both in the case of normal and suppressed (by 99%) replication by arabofuranosylcytosine [3H]-thymidine and [3H-5-methyl] cytosine are detected in the Okazaki fragments (5S) as well as in some discrete lower molecular weight fractions (lesser than 5S) of newly synthesized DNA. Thus, replicative methylation of DNA in the fibroblasts occurs in the replicative fork during DNA synthesis and the functioning DNA methylase is an indispensable component of the replicative complex. The methylation of Okazaki fragments is non-chaotic and has a specificity other than that of total DNA. This may be due to the multiplicity and different specificity of nuclear DNA-methylases. Thus, there exist in animal cells replicative and post-replicative methylation of DNA, which may differ in the nature of substrates and enzymes, in specificity of recognizable sequences and in their functional significanse.     

Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin

We investigated concerted evolution of rRNA genes in multiple populations of Tragopogon mirus and T. miscellus, two allotetraploids that formed recurrently within the last 80 years following the introduction of three diploids (T. dubius, T. pratensis, and T. porrifolius) from Europe to North America. Using the earliest herbarium specimens of the allotetraploids (1949 and 1953) to represent the genomic condition near the time of polyploidization, we found that the parental rDNA repeats were inherited in roughly equal numbers. In contrast, in most present-day populations of both tetraploids, the rDNA of T. dubius origin is reduced and may occupy as little as 5% of total rDNA in some individuals. However, in two populations of T. mirus the repeats of T. dubius origin outnumber the repeats of the second diploid parent (T. porrifolius), indicating bidirectional concerted evolution within a single species. In plants of T. miscellus having a low rDNA contribution from T. dubius, the rDNA of T. dubius was nonetheless expressed. We have apparently caught homogenization of rDNA repeats (concerted evolution) in the act, although it has not proceeded to completion in any allopolyploid population yet examined.     

Generality and characteristics of genetic and epigenetic changes in newly synthesized allotetraploid wheat lines

Previous studies have shown rapid and extensive genomic instability associated with early stages of allopolyploidization in wheat.However, these studies are based on either a few pre-selected genomic loci or genome-wide analysis of a single plant individual for a given cross combination, thus making the extent and generality of the changes uncertain.To further study the generality and characteristics of allopolyploidization-induced genomic instability in wheat, we investigated genetic and epigenetic changes from a genome-wide perspective (by using the AFLP and MSAP markers) in four sets of newly synthesized allotetraploid wheat lines with various genome constitutions, each containing three randomly chosen individual plants at the same generation.We document that although general chromosomal stability was characteristic of all four sets of allotetraploid wheat lines, genetic and epigenetic changes at the molecular level occurred in all these plants, with both kinds of changes classifiable into two distinct categories, i.e., stochastic and directed.The abundant type of genetic change is loss of parental bands while the prevalent cytosine methylation pattern alteration is hypermethylation at the CHG sites.Our results have extended previous studies regarding allopolyploidization-induced genomic dynamics in wheat by demonstrating the generality of both genetic and epigenetic changes associated with multiple nascent allotetraploid wheat lines, and providing novel insights into the characteristics of the two kinds of induced genomic instabilities.     

Biological evaluation of newly synthesized aryl(thio)carbamoyl derivatives of 1- and 1-(2-aminoethyl)-piperazines

Twelve 1-methyl and acetyl-4-substituted piperazines, evaluated as potential herbicides and plant growth regulators, were synthesized by condensation of 1-methyl-piperazine and 1-[2-(acetylamino)ethyl]-4-acetyl-piperazine with the corresponding aryliso(thio)cyanates. These piperazines, which incorporate a piperazine ring and aryl(thio)carbamoyl groups connected directly or through an ethylene group, are new chemical families of herbicides and cytokinin mimics. Structure–activity relationships for the screened compounds were evaluated and discussed. The greatest herbicidal activity against Triticum aestivum was observed with compounds that contained the three structural elements: piperazine ring, ethylene group and 4-fluorophenylcarbamoyl group. Compounds having a combination of two active moieties–piperazine ring and 4-halogenophenylthiocarbamoyl group, also showed high herbicidal activity against T. aestivum. The compound, in which the un-substituted phenylcarbamoyl group was directly connected to the piperazine ring, showed cytokinin-like activity and significantly stimulated betacyanin synthesis in Amaranthus caudatus.     

Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion

To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G2/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor.     

Visual verification of close disposition between a rice A genome-specific DNA sequence (TrsA) and the telomere sequence

A rice A genome-specific tandem repeat sequence (TrsA) and telomeric nucleotide sequences, (TTTAGGG)_n, were simultaneously detected by multicolor fluorescence in situ hybridization (McFISH) using rice prometaphase chromosomes. Six pairs of TrsA sites visualized by fluorescence signals were all localized on the long arms close to the telomeric regions. Differences in the copy number of TrsA at the different sites were visualized both by the size of the telomeric condensation block stained with Giemsa solution and the signal intensity after FISH with TrsA. McFISH analyses using interphase nuclei could resolve close disposition of TrsA and telomere and also gave rough estimation of the distance between them. The functional significance of the close disposition of TrsA and telomere is discussed.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.