RESUMO
Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína de Replicação A/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Reparo do DNA/fisiologia , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genéticaRESUMO
BACKGROUND: The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported. RESULTS: We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted. CONCLUSIONS: Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA de Plantas/genética , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , DNA de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Plants have evolved a unique epigenetic process to target DNA cytosine methylation: RNA-directed DNA methylation (RdDM). During RdDM, small RNAs (smRNAs) guide methylation of homologous DNA loci. In Arabidopsis thaliana, the de novo DNA methyltransferase that ultimately methylates cytosines guided by smRNAs in all sequence contexts is Domains Rearranged Methyltransferase 2 (DRM2). Recent reports have shown that DRM2 requires the catalytic mutated paralog DRM3 to exert its function through a still largely unknown process. To shed light on how DRM3 affects RdDM, we have further characterized its role at the molecular and cytological levels. FINDINGS: Although DRM3 is not required for RdDM loci transcriptional silencing, it specifically affects loci's DNA methylation. Interestingly, DRM3 and DRM2 regulate the DNA methylation in a subset of loci differently.Fluorescence In Situ Hybridization and immunolocalization analyses showed that DRM3 is not required for the large-scale nuclear organization of heterochromatin during interphase, with the notable exception of the 45S ribosomal RNA loci. DRM3 localizes exclusively to the nucleus and is enriched in a round-shaped domain located in the nucleolar periphery, in which it colocalizes with components of the RdDM pathway. CONCLUSIONS: Our analyses reinforce the previously proposed chaperone role of DRM3 in RdDM. Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases. However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent. DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.
Assuntos
Arabidopsis/genética , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , RNA de Plantas/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Nucléolo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Metiltransferases/metabolismo , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Interferência de RNA , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
Noncoding RNAs are the rising stars of genome regulation and are crucial to an organism's metabolism, development, and defense. One of their most notable functions is its ability to direct epigenetic modifications through small RNA molecules to specific genomic regions, ensuring transcriptional regulation, proper genome organization, and maintenance of genome integrity. Here, we review the current knowledge of the spatial organization of the Arabidopsis thaliana RNA-directed DNA methylation pathway within the cell nucleus, which, while known to be essential for the proper establishment of epigenetic modifications, remains poorly understood. We will also discuss possible future cytological approaches that have the potential of unveiling functional insights into how small RNA-directed epigenetics is regulated through the spatiotemporal regulation of its major components within the cell.
Assuntos
Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , Epigênese Genética , Interferência de RNA , RNA não Traduzido/genética , RNA não Traduzido/isolamento & purificação , Análise de Sequência de DNARESUMO
In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.
Assuntos
Arabidopsis/genética , Corpos Enovelados/metabolismo , MicroRNAs/metabolismo , Proteínas de Plantas/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas de Arabidopsis/metabolismo , Western Blotting , Primers do DNA/genética , Imunofluorescência , MicroRNAs/biossíntese , Microscopia de Fluorescência , Plantas Geneticamente Modificadas/genética , RNA Interferente Pequeno/biossíntese , Ribonuclease III/metabolismoRESUMO
Plant specific SGS3-like proteins are composed of various combinations of an RNA-binding XS domain, a zinc-finger zf-XS domain, a coil-coil domain and a domain of unknown function called XH. In addition to being involved in de novo 2 (IDN2) and SGS3, the Arabidopsis genome encodes 12 uncharacterized SGS3-like proteins. Here, we show that a group of SGS3-like proteins act redundantly in RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. Transcriptome co-expression analyses reveal significantly correlated expression of two SGS3-like proteins, factor of DNA methylation 1 (FDM1) and FDM2 with known genes required for RdDM. The fdm1 and fdm2 double mutations but not the fdm1 or fdm2 single mutations significantly impair DNA methylation at RdDM loci, release transcriptional gene silencing and dramatically reduce the abundance of siRNAs originated from high copy number repeats or transposons. Like IDN2 and SGS3, FDM1 binds dsRNAs with 5' overhangs. Double mutant analyses also reveal that IDN2 and three uncharacterized SGS3-like proteins FDM3, FDM4 and FDM5 have overlapping function with FDM1 in RdDM. Five FDM proteins and IDN2 define a group of SGS3-like proteins that possess all four-signature motifs in Arabidopsis. Thus, our results demonstrate that this group of SGS3-like proteins is an important component of RdDM. This study further enhances our understanding of the SGS3 gene family and the RdDM pathway.
Assuntos
Proteínas de Arabidopsis/fisiologia , Metilação de DNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mutação , RNA Polimerase II/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA não Traduzido/biossíntese , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.
Assuntos
Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , DNA Polimerase II/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de RNAr/genética , Histona Desacetilases/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Arabidopsis/genética , DNA Intergênico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Histona Desacetilases/genética , Histonas/metabolismo , Metilação , MutaçãoRESUMO
Nucleolar dominance is a widespread epigenetic phenomenon, describing the preferential silencing of ribosomal RNA (rRNA) genes inherited from one progenitor of an interspecific hybrid, independent of maternal or paternal effects. In the allotetraploid hybrid plant species Arabidopsis suecica, A. thaliana-derived rRNA genes are silenced whereas the A. arenosa-derived rRNA genes are transcribed. We reported previously on an RNAi-based screen of DNA methyltransferases, methylcytosine binding proteins and RNA-dependent DNA methylation pathway proteins that identified specific activities required for the establishment or enforcement of nucleolar dominance. Here we present additional molecular and cell biological evidence that siRNA-directed cytosine methylation and the methylcytosine binding protein MBD6 bring about large-scale chromosomal effects on rRNA gene loci subjected to nucleolar dominance in A. suecica.
Assuntos
Proteínas de Arabidopsis/metabolismo , Nucléolo Celular/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Heterocromatina/metabolismo , RNA de Plantas/genética , RNA Ribossômico/genética , Transporte Ativo do Núcleo Celular , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Citosina/metabolismo , Hibridização Genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , RNA de Plantas/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs (siRNAs) are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and DICER-LIKE 3 (DCL3) are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 (AGO4). Nuclear RNA polymerase V (Pol V) collaborates with DRD1 (DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1) to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drd1 mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Heterocromatina/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Imunofluorescência , Regulação da Expressão Gênica de Plantas , Hibridização In Situ , Microscopia de Fluorescência , RNA Interferente Pequeno/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de SinaisRESUMO
In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2, and the methylcytosine binding domain proteins, MBD6 and MBD10, as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Nucléolo Celular/genética , Citosina/metabolismo , Metilação de DNA , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Pareamento de Bases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Intergênico , Heterocromatina/metabolismo , Modelos Biológicos , Região Organizadora do Nucléolo/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Interferência de RNA , RNA de Plantas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana -derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.
Assuntos
Nucléolo Celular/genética , Inativação Gênica , Genes de Plantas , Arabidopsis/embriologia , Arabidopsis/genética , Sequência de Bases , Primers do DNA , Região Organizadora do Nucléolo , RNA Ribossômico/genéticaRESUMO
B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.
Assuntos
Cromossomos de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Heterocromatina/genética , Secale/genética , Transcrição Gênica , Núcleo Celular/metabolismo , DNA de Plantas/metabolismo , Genoma de Planta , Histonas/metabolismo , Interfase , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Especificidade de Órgãos , Poliadenilação , Sequências Repetitivas de Ácido Nucleico , Secale/citologia , Secale/crescimento & desenvolvimentoRESUMO
In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.
Assuntos
Arabidopsis/genética , Nucléolo Celular/genética , Cromatina/genética , DNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA/biossíntese , RNA/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Metilação de DNA , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Inativação Gênica/fisiologia , Substâncias Macromoleculares/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais/genéticaRESUMO
All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.
Assuntos
Arabidopsis/enzimologia , Núcleo Celular/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Heterocromatina/enzimologia , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico/genética , Núcleo Celular/genética , Metilação de DNA , RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Heterocromatina/genética , Dados de Sequência Molecular , Mutação/genética , Filogenia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , tRNA Metiltransferases/metabolismoRESUMO
'Lindström' wheat (AABBDD+rye B chromosomes) was used to study the effects of alien chromatin introgressed into a wheat genetic background, subjecting the wheat genome to a new and transient allopolyploidisation episode. Using this experimental material, we have previously demonstrated that no large-scale chromosomal translocations occurred as a result of the genomic constitution of the addition line. However, we have shown that the presence of a number of rye B chromosomes is associated with changes in the interphase organization and expression patterns of wheat rDNA loci. We have now extended our studies to focus on a further characterization of 'Lindström' 5S rDNA loci and also on high molecular weight glutenin subunit (HMW-GS) patterns. In the process, we have uncovered an unusually large variant of the 5S rDNA locus on wheat chromosome 1B (not to be confused with rye B chromosomes) and 2 novel HMW glutenin y-type alleles. These changes are not directly related to variation in rye B chromosome number in the present material, but the fact that a new, and still segregating, 1Dy HMW-GS gene was identified indicates a recent timescale for its origin. Strikingly, the 'Lindström' 5S rDNA 1B locus integrates a unit sharing 94% homology with a rye 5S rDNA sequence, suggesting the possibility that the wheat locus was colonized by highly homologous rye sequences during the breeding of 'Lindström', when the rye and wheat genomes were together, albeit briefly, in the same nucleus.