Chromatin proteins and RNA are associated with DNA during all phases of mitosis.

Mitosis brings about major changes to chromosome and nuclear structure. We used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. We also found that RNA transcripts are associated with DNA during all stages of mitosis. Reduction of H3K27me3 levels or elimination of RNAs had no effect on the association of the components of PcG and TrxG complexes to DNA. Using a combination of proximity ligation assay-based techniques and super-resolution microscopy, we found that the number of protein-DNA and RNA-DNA foci undergoes significant reduction during mitosis, suggesting that mitosis may be accompanied by structural re-arrangement or compaction of specific chromatin domains

Characterization of the Arabidopsis thaliana SCRc and SRKc Pseudogenes via Reporter Gene Analysis

Plants in the family Brassicaceae have evolved a mechanism for self-incompatibility (SI) that is initiated by two genes encoded at the Sterility (S)-locus: S-locus Cysteine Rich (SCR) and S-locus Receptor Kinase (SRK). The self-compatible species Arabidopsis thaliana has lost its capacity to activate SI, potentially due to the nonfunctionality and/or altered expression of the SCR and SRK genes. The C haplotype, one of the three S-locus haplotypes remaining in A. thaliana, was recently identified as having an SRK that encodes a nonfunctional protein product, but an SCR that encodes a functional protein (Dwyer et al., 2013). Real-time PCR was used to ascertain that both AtSCRc and AtSRKc synthesized lower levels of transcripts compared with functional SCR and SRK genes from self-incompatible Arabidopsis lyrata. The study performed here utilized the reporter gene β-glucuronidase (GUS) in order to assess the tissue-specific and developmental-specific expression of AtSCRc and AtSRKc. Transgenes were constructed wherein each putative regulatory region upstream of either pseudogene (designated AtSCRCp or AtSRKCp) was inserted into the Ti reporter gene vector pBI101 and subsequently were transformed into A. thaliana plants by Agrobacteria. Transgenic plant tissue, especially floral tissue, was collected and histochemically stained with an X-gluc solution which turned blue in regions of the plant that expressed GUS. AtSCRCp::GUS showed expression in immature to mature anthers (as expected). AtSRKCp::GUS, however, demonstrated ectopic expression in the subpapillar cell region of pistils, in immature anthers, or in a combination of both of these regions (but not in the papillar cells, as expected for a functional SRK gene). Further analyses were performed by comparison of the AtSCRCp and AtSRKCp nucleotide sequences with the upstream regions of other functionally expressed SCR and SRK genes in hopes of delineating potential regulatory motifs associated with these two genes