Vratislav Peška

Two faces of Solanaceae telomeres: a comparison between Nicotiana and Cestrum telomeres and telomere-binding proteins

While most Solanaceae genera (e.g.Solanum, Nicotiana) possess Arabidopsis-type telomeres of (TTTAGGG)n maintained by telomerase, the genera Cestrum, Vestia and Sessea (Cestrum group) lack these telomeres. Here we show that in the Cestrum-group the activity of telomerase has been lost. Nevertheless, proteins binding the single-stranded G-rich strand of the Arabidopsis-type and related human-type (TTAGGG)n telomeric sequences are present in nuclear extracts of both Nicotiana and Cestrum species. These proteins may have a role in telomere function or other cellular activities. In addition to characterizing DNA binding specificity and molecular weights of these proteins, we searched in both N. tabacum (tobacco) and C. parqui for the presence of POT1-like proteins, involved in telomere capping and telomerase regulation. Analysis of POT1-like proteins available on public databases and cloned by us from C. parqui, revealed the N-terminal OB folds typical for this protein family and a novel, plant-specific conserved C-terminal OB-fold domain (CTOB). We propose that CTOB is involved in protein-protein interactions.

Structure-function relationships during transgenic telomerase expression in Arabidopsis

Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

Replication of ribosomal DNA in Arabidopsis occurs both inside and outside the nucleolus during S phase progression

ABSTRACT Ribosomal RNA genes (rDNA) have been used as valuable experimental systems in numerous studies. Here, we focus on elucidating the spatiotemporal organisation of rDNA replication in Arabidopsis thaliana. To determine the subnuclear distribution of rDNA and the progression of its replication during the S phase, we apply 5-ethynyl-2′-deoxyuridine (EdU) labelling, fluorescence-activated cell sorting, fluorescence in situ hybridization and structured illumination microscopy. We show that rDNA is replicated inside and outside the nucleolus, where active transcription occurs at the same time. Nascent rDNA shows a maximum of nucleolar associations during early S phase. In addition to EdU patterns typical for early or late S phase, we describe two intermediate EdU profiles characteristic for mid S phase. Moreover, the use of lines containing mutations in the chromatin assembly factor-1 gene fas1 and wild-type progeny of fas1xfas2 crosses depleted of inactive copies allows for selective observation of the replication pattern of active rDNA. High-resolution data are presented, revealing the culmination of replication in the mid S phase in the nucleolus and its vicinity. Taken together, our results provide a detailed snapshot of replication of active and inactive rDNA during S phase progression. Summary: In Arabidopsis, replication of active and inactive rDNA occurs during S phase progression. High-resolution data reveal the culmination of replication in the mid S phase in the nucleolus.

A polymerase chain reaction-based approach for evaluation of telomere-associated sequences and interstitial telomeric sequences

Telomere minisatellites could be present in both terminal and internal chromosomal regions. We monitored the progress of BAL-31 nuclease digestion on Arabidopsis thaliana genomic DNA prepared by standard isolation techniques to verify its cleavage at terminal and internal genomic regions. A subtelomeric position of candidate sequences was validated using conventional polymerase chain reaction (PCR), combining the C-strand-specific telomeric primer with a subtelomeric reverse primer, and confirmed by quantitative PCR (qPCR) using sequence-specific primer pairs on DNA samples after BAL-31 digestion. qPCR amplification showed a gradual decrease in subtelomeric sequence signals, in contrast to interstitial telomeric sequences from pericentromere and control sequences.