Jana Fulnečková

Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths

Abstract Changes in telomere lengths and telomerase activity in tobacco cells were studied during dedifferentiation and differentiation; leaf tissues were used to initiate callus cultures, which were then induced to regenerate plants. While no significant changes in the range of telomere lengths were observed in response to dedifferentiation and differentiation, there was a conspicuous increase in telomerase activity in calli compared to the source leaves, where the activity was hardly detectable. In leaves of regenerated plants, the telomerase activity fell to almost the same level as in the original plant, showing on the average 0.04% of the level in callus. The process was then repeated using the regenerants as the source material. In the second round of dedifferentiation and differentiation, telomerase activity showed a similar increase in calli derived from regenerated plants and a drop in plants regenerated from these calli. Telomere lengths remained unchanged both in calli and in leaves of regenerants. The conservation of telomere lengths over repeated rounds of dedifferentiation and differentiation, which are associated with dramatic changes in cell division rate and corresponding variation in telomerase activity may reflect the function of a regulatory mechanism in plant cells which controls telomerase action to compensate for replicative loss of telomeric DNA.

Adsorptive Transfer Stripping AC Voltammetry of DNA Complexes with Intercalators

Using alternating current adsorptive transfer stripping voltammetry at hanging mercury drop electrode (HMDE), conformational changes of DNA due to binding of DNA intercalators were studied by means of the measurements of tensammetric DNA signals of peak 2 and peak 3. Untwisting of DNA by the intercalators in solution resulted in an altered DNA adsorption. After medium exchange and intercalator removal, the surface-confined DNA probably adopted a restrained structure, with untwisted segments attached to the electrode surface (yielding peak 2) and superhelical loops extending to the bulk of solution. This structure was more resistant to DNA surface denaturation than dsDNA adsorbed at HMDE in the absence of intercalators, which resulted in a decreased intensity of DNA peak 3. Similar effects were also observed when DNA was adsorbed at the electrode from a solution of low salt concentration. Upon introduction of DNA single-strand breaks, the specific behavior of DNA-intercalator complexes was eliminated.

Inhibition of plant telomerase by telomere-binding proteins from nuclei of telomerase-negative tissues

The activity of telomerase in plant cells is precisely regulated in response to changes in cell division rate. To explore this regulatory mechanism, the effect on telomerase activity of protein extracts from nuclei of telomerase‐negative tissues was examined. An inhibition of telomerase activity was found which was species‐non‐specific. This inhibition was due to proteins which form salt‐stable, sequence‐specific complexes with the G‐rich telomeric strand and reduce its accessibility, as shown by gel retardation and by terminal transferase (TdT) extension of G‐rich telomeric and non‐telomeric (substrate) primers. A 40 kDa polypeptide was detected by SDS–PAGE after cross‐linking the complex formed by extracts from tobacco leaf nuclei. Such proteins may be involved in regulation of telomerase activity in plants.

A Broad Phylogenetic Survey Unveils the Diversity and Evolution of Telomeres in Eukaryotes

Telomeres, ubiquitous and essential structures of eukaryotic chromosomes, are known to come in a variety of forms, but knowledge about their actual diversity and evolution across the whole phylogenetic breadth of the eukaryotic life remains fragmentary. To fill this gap, we employed a complex experimental approach to probe telomeric minisatellites in various phylogenetically diverse groups of algae. Our most remarkable results include the following findings: 1) algae of the streptophyte class Klebsormidiophyceae possess the Chlamydomonas-type telomeric repeat (TTTTAGGG) or, in at least one species, a novel TTTTAGG repeat, indicating an evolutionary transition from the Arabidopsis-type repeat (TTTAGGG) ancestral for Chloroplastida; 2) the Arabidopsis-type repeat is also present in telomeres of Xanthophyceae, in contrast to the presence of the human-type repeat (TTAGGG) in other ochrophytes studied, and of the photosynthetic alveolate Chromera velia, consistent with its phylogenetic position close to apicomplexans and dinoflagellates; 3) glaucophytes and haptophytes exhibit the human-type repeat in their telomeres; and 4) ulvophytes and rhodophytes have unusual telomere structures recalcitrant to standard analysis. To obtain additional details on the distribution of different telomere types in eukaryotes, we performed in silico analyses of genomic data from major eukaryotic lineages, utilizing also genome assemblies from our on-going genome projects for representatives of three hitherto unsampled lineages (jakobids, malawimonads, and goniomonads). These analyses confirm the human-type repeat as the most common and possibly ancestral in eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages, some of them several times independently.

Dynamic Evolution of Telomeric Sequences in the Green Algal Order Chlamydomonadales

Telomeres, which form the protective ends of eukaryotic chromosomes, are a ubiquitous and conserved structure of eukaryotic genomes but the basic structural unit of most telomeres, a repeated minisatellite motif with the general consensus sequence TnAmGo, may vary between eukaryotic groups. Previous studies on several species of green algae revealed that this group exhibits at least two types of telomeric sequences, a presumably ancestral type shared with land plants (Arabidopsis type, TTTAGGG) and conserved in, for example, Ostreococcus and Chlorella species, and a novel type (Chlamydomonas type, TTTTAGGG) identified in Chlamydomonas reinhardtii. We have employed several methodical approaches to survey the diversity of telomeric sequences in a phylogenetically wide array of green algal species, focusing on the order Chlamydomonadales. Our results support the view that the Arabidopsis-type telomeric sequence is ancestral for green algae and has been conserved in most lineages, including Mamiellophyceae, Chlorodendrophyceae, Trebouxiophyceae, Sphaeropleales, and most Chlamydomonadales. However, within the Chlamydomonadales, at least two independent evolutionary changes to the Chlamydomonas type occurred, specifically in a subgroup of the Reinhardtinia clade (including C. reinhardtii and Volvox carteri) and in the Chloromonadinia clade. Furthermore, a complex structure of telomeric repeats, including a mix of the ancestral Arabidopsis-type motifs and derived motifs identical to the human-type telomeric repeats (TTAGGG), was found in the chlamydomonadalean clades Dunaliellinia and Stephanosphaeria. Our results indicate that telomere evolution in green algae, particularly in the order Chlamydomonadales, is far more dynamic and complex than thought before. General implications of our findings for the mode of telomere evolution are discussed.

Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis.

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.

Evolutionarily Distant Streptophyta Respond Differently to Genotoxic Stress

Research in algae usually focuses on the description and characterization of morpho—and phenotype as a result of adaptation to a particular habitat and its conditions. To better understand the evolution of lineages we characterized responses of filamentous streptophyte green algae of the genera Klebsormidium and Zygnema, and of a land plant—the moss Physcomitrella patens—to genotoxic stress that might be relevant to their environment. We studied the induction and repair of DNA double strand breaks (DSBs) elicited by the radiomimetic drug bleomycin, DNA single strand breaks (SSB) as consequence of base modification by the alkylation agent methyl methanesulfonate (MMS) and of ultra violet (UV)-induced photo-dimers, because the mode of action of these three genotoxic agents is well understood. We show that the Klebsormidium and Physcomitrella are similarly sensitive to introduced DNA lesions and have similar rates of DSBs repair. In contrast, less DNA damage and higher repair rate of DSBs was detected in Zygnema, suggesting different mechanisms of maintaining genome integrity in response to genotoxic stress. Nevertheless, contrary to fewer detected lesions is Zygnema more sensitive to genotoxic treatment than Klebsormidium and Physcomitrella