The promoter homology-dependent inactivation of a 35Spro-hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady-state levels of Hyg and NiR transcripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non-uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post-transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by "epigene conversion" involving DNA-DNA pairing.

Chinese cabbage plants remain in the vegetative growth phase until they have experienced prolonged exposure to cold temperature, known as vernalization. This inhibition of flowering is caused by the high levels of FLOWERING LOCUS C (FLC) expression. To increase the product value of Chinese cabbage by inhibiting the floral transition, three genes (BrFLC1, BrFLC2, and BrFLC3) homologous to the AtFLC gene, which encodes a floral repressor, were isolated from the Chinese cabbage 'Chiifu'. These genes showed high similarity to AtFLC, although the putative BrFLC1 protein contained ten more residues than AtFLC. The BrFLC genes were expressed ubiquitously, except that BrFLC3 was not expressed in roots. BrFLC1 and BrFLC2 showed stronger expression than BrFLC3 in unvernalized and vernalized Chinese cabbage. The expression levels of the three BrFLC genes were lower in an early-flowering Chinese cabbage, suggesting that the BrFLC transcript level was associated with flowering time. Constitutive expression of the BrFLC genes in Arabidopsis significantly delayed flowering, which was also observed in transgenic Chinese cabbage overexpressing BrFLC3. These results suggest that the BrFLC genes act similarly to AtFLC. Our results provide a technique for controlling flowering time in Chinese cabbage and other crops to produce high yields of vegetative tissues.

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In this study, the microstructural characterisation, mechanical testing and fractography investigation were performed on twinning induced plasticity (TWIP) steels, fabricated with resistance spot welding. Failure mode during the cross-tensile test was found to follow the sequences of strain localisation of both sheets, crack initiation at notch tip, crack following along the fusion boundary and, finally, ductile shear fracture along the sheet thickness direction. On the other hand, failure in the tensile shear test was always directed along the sheet/sheet (s/s) interface; the interfacial failure and shear deformation were observed at the weld centreline. Solidification occurred as a primary austenitic solidification mode, and no martensitic transformations were detected through electron backscatter diffraction analysis. The fusion zone was mainly composed of austenite with directional solidification towards the centreline; the columnar dendritic and equiaxed structures were identified. Interdendritic C and Mn cosegregation were confirmed by electron probe microanalysis.

Five furanocoumarins including a new one were isolated from the root of Angelica dahurica by repeated silica gel column chromatography. Their chemical structures were determined to be isoimperatorin (1), oxypeucedanin hydrate-3"-butyl ether (2), imperatorin (3), knidilin (4), and oxypeucedanin hydrate (5). This represents the first study in which the compound 2 has been isolated and identified. The long-range coupling (5J) in the 1H-NMR spectrum observed in the linear furanocoumarin skeleton was also investigated in detail.

To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage ( Brassica rapa ssp. pekinensis ), floral bud transcriptome analysis was carried out using a B . rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B . rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.

This paper describes molecular and cytogenetic evidence for the stability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro, suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcinogenesis are discussed.