T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites

A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2–3 base pairs long, that preferentially includes a G close to the right end of the T-DNA.     

Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome.

Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

Conservation and progressive methylation of Epstein-Barr viral DNA sequences in transformed cells.

The structure of intracellular viral DNA from a number of cell lines arising by clonal transformation of human lymphocytes in vitro with Epstein-Barr virus was analyzed. Intracellular viral DNAs were partially purified and digested with several restriction endonucleases, and the products of digestion were separated by electrophoresis in agarose gels. The viral fragments were detected by transferring the DNA from the gel to nitrocellulose sheets, hybridizing radiolabeled recombinant vectors carrying fragments of viral DNA to those transfers, and visualizing the hybrids by autoradiography. These analyses indicated that: (i) regions of repetitious viral DNA do undergo expansion and contraction although one size predominates; (ii) novel sequence arrangements appear in the intracellular viral DNA of different clones but are not found in clones analyzed serially and propagated extensively; (iii) the viral DNA is increasingly methylated upon cell propagation. We have not identified a transformed cell phenotype or a viral phenotype that segregates with the observed progressive methylation. We have not detected in Epstein-Barr viral plasmids analogs of the gross rearrangements of viral DNAs observed after lytic infections with high multiplicities of papova-, adeno-, or herpes simplex viruses.     

Cytological characterization of the tandem repetitive sequences and their methylation status in the Antirrhinum majus genome

Tandem repetitive sequences are DNA motifs common in the genomes of eukaryotic species and are often embedded in heterochromatic regions. In most eukaryotes, ribosomal genes, as well as centromeres and telomeres or subtelomeres, are associated with abundant tandem arrays of repetitive sequences and typically represent the final barriers to completion of whole-genome sequencing. The nature of these repeats makes it difficult to estimate their actual sizes. In this study, combining the two cytological techniques DNA fiber-FISH and pachytene chromosome FISH allowed us to characterize the tandem repeats distributed genome wide in Antirrhinum majus and identify four types of tandem repeats, 45S rDNA, 5S rDNA, CentA1, and CentA2, representing the major tandem repetitive components, which were estimated to have a total length of 18.50 Mb and account for 3.59% of the A. majus genome. FISH examination revealed that all the tandem repeats correspond to heterochromatic knobs along the pachytene chromosomes. Moreover, the methylation status of the tandem repeats was investigated in both somatic cells and pollen mother cells from anther tissues using an antibody against 5-methylcytosine combined with sequential FISH analyses. Our results showed that these repeats were hypomethylated in anther tissues, especially in the pollen mother cells at pachytene stage.     

Costs of resistance to fungal pathogens in genetically modified wheat

Aims Many resistance genes against fungal pathogens show costs of resistance. Genetically modified (GM) plants that differ in only one or a few resistance genes from control plants present ideal systems for measuring these costs in the absence of pathogens.Methods To assess the ecological relevance of costs of pathogen resistance, we grew individual plants of four transgenic spring wheat lines in a field trial with three pathogen levels and varied the genetic diversity of the crop.Important findings We found that two lines with a Pm3b transgene were more resistant to powdery mildew than their sister lines of the variety Bobwhite, whereas lines with chitinase (A9) or chitinase and glucanase (A13) transgenes were not more resistant than their mother variety Frisal. Nevertheless, in the absence of the pathogen, both the GM lines of Bobwhite as well as those of Frisal performed significantly worse than their controls, i.e. Pm3b #1 and Pm3b #2 had 39% or 53% and A9 and A13 had 14% or 23% lower yields. In the presence of the pathogen, all GM lines except Pm3b #2 could increase their yields and other fitness-related traits, reaching the performance levels of the control lines. Line Pm3b #2 seemed to have lost its phenotypic plasticity and had low performance in all environments. This may have been caused by very high transgene expression. No synergistic effects of mixing different GM lines with each other were detected. This might have been due to high transgene expression or the similarity between the lines regarding their resistance genes. We conclude that costs of resistance can be high for transgenic plants with constitutive transgene expression and that this can occur even in cases where the non-transgenic control lines are already relatively resistant, such as in our variety Frisal. Transgenic plants could only compete with conventional varieties in environments with high pathogen pressure. Furthermore, the large variability among the GM lines, which may be due to unpredictable transgene expression, suggests that case-by-case assessments are necessary to evaluate costs of resistance.     

Clarifying the regulation of genome editing in Australia: situation for genetically modified organisms

Australia’s gene technology regulatory scheme (GT Scheme) regulates activities with genetically modified organisms (GMOs, organisms modified by gene technology), including environmental releases. The scope of regulation, i.e. what organisms are and are not regulated, is set by the Gene Technology Act 2000 (GT Act) and GT Regulations 2001 (GT Regulations). The GT Act gives broad, overarching definitions of ‘gene technology’ and ‘GMO’ but also provides for exclusions and inclusions in the GT Regulations. Whether organisms developed with genome editing techniques are, or should be, regulated under countries’ national GMO laws is the subject of debate globally. These issues are also under active consideration in Australia. A technical review of the GT Regulations was initiated in 2016 to clarify the regulatory status of genome editing. Proposed draft amendments are structured around whether the process involves introduction of a nucleic acid template. If agreed, amendments would exclude from regulation organisms produced using site directed nuclease (SDN) 1 techniques while organisms produced using oligonucleotide mutagenesis, SDN-2 or SDN-3 would continue to be regulated as GMOs. The review of the GT Regulations is still ongoing and no legislative changes have been made to the GT Regulations. A broader policy review of the GT Scheme was undertaken in 2017–2018 and as a result further work will be undertaken on the scope and definitions of the GT Act in light of ongoing developments.

     

Regulation beyond genome sequences: DNA and histone methylation in embryonic stem cells

Embryonic stem (ES) cells distinct themselves from other cell type populations by their pluripotent ability. The unique features of ES cells are controlled by both genetic and epigenetic factors. Studies have shown that the methylation status of DNA and histones in ES cells is quite different from that of differentiated cells and somatic stem cells. Herein, we summarized recent advances in DNA and histone methylation studies of mammalian ES cells. The methylation status of several key pluripotent regulatory genes is also discussed.     

Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast

Despite increasing awareness of the importance of the mitochondrial genome in human pathology, very few attempts have been made so far toward genetic engineering of mitochondrial DNA (mtDNA). One of the reasons for this slow progress is the difficulty of cloning mtDNA in Escherichia coli, a trait in common with repetitive or palindromic sequences, and some viral sequences. We have previously made a construct containing the entire mouse mitochondrial genome and a cDNA sequence coding for human ornithine transcarbamylase in a yeast/bacterial shuttle vector, which can be stably maintained in E. coli. We wished to modify this vector for mitochondrial gene therapy by the addition of mitochondrial chloramphenicol resistance, conferred by a point mutation in the 16S rRNA gene. Attempts to modify this construct by a straightforward cloning approach in E. coli proved unsuccessful. Two successful strategies for modification of large unstable constructs in both E. coli and the yeast Saccharomyces cerevisiae are compared here.     

T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation

Transgenic Arabidopsis and tobacco plants (125) derived from seven Agrobacterium-mediated transformation experiments were screened by polymerase chain reaction and DNA gel blot analysis for the presence of vector `backbone' sequences. The percentage of plants with vector DNA not belonging to the T-DNA varied between 20% and 50%. Neither the plant species, the explant type used for transformation, the replicon type nor the selection seem to have a major influence on the frequency of vector transfer. Only the border repeat sequence context could have an effect because T-DNA vector junctions were found in more than 50% of the plants of three different transformation series in which T-DNAs with octopine borders without inner border regions were used. Strikingly, many transgenic plants contain vector backbone sequences linked to the left T-DNA border as well as vector junctions with the right T-DNA border. DNA gel blots indicate that in most of these plants the complete vector sequence is integrated. We assume that integration into the plant genome of complete vector backbone sequences could be the result of a conjugative transfer initiated at the right border and subsequent continued copying at the left and right borders, called read-through. This model would imply that the left border is not frequently recognized as an initiation site for DNA transfer and that the right border is not efficiently recognized as a termination site for DNA transfer.     

Germline methylation patterns inferred from local nucleotide frequency of repetitive sequences in the human genome

Given the genomic abundance and susceptibility to DNA methylation, interspersed repetitive sequences in the human genome can be exploited as valuable resources in genome-wide methylation studies. To learn about the relationships between DNA methylation and repeat sequences, we performed a global measurement of CpG dinucleotide frequencies for interspersed repetitive sequences and inferred germline methylation patterns in the human genome. Although extensive CpG depletion was observed for most repeat sequences, those in the proximity to CpG islands have been relatively removed from germline methylation being the potential source of germline activation. We also investigated the CpG depletion patterns of Alu pairs to see whether they might play an active role in germline methylation. Two kinds of Alu pairs, direct or inverted pairs classified according to the orientation, showed contrast CpG depletion patterns with respect to separating distance of Alus, i.e., as two Alu elements are more closely spaced in a pair, a higher extent of CpG depletion was observed in inverted orientation and vice versa for directly repetitive Alu pairs. This suggests that specific organization of repetitive sequences, such as inverted Alu pairs, might play a role in triggering DNA methylation consistent with a homology-dependent methylation hypothesis.     

A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome

A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected. When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.     

Expression of bar in the plastid genome confers herbicide resistance

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.     

Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library

We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes.