Jiří Fajkus

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.

Telomeres in evolution and evolution of telomeres

This paper examines telomeres from an evolutionary perspective. In the monocot plant order Asparagales two evolutionary switch-points in telomere sequence are known. The first occurred when the Arabidopsis-type telomere was replaced by a telomere based on a repeat motif more typical of vertebrates. The replacement is associated with telomerase activity, but the telomerase has low fidelity and this may have implications for the binding of telomeric proteins. At the second evolutionary switch-point, the telomere and its mode of synthesis are replaced by an unknown mechanism. Elsewhere in plants (Sessia, Vestia, Cestrum) and in arthropods, the telomere “typical” of the group is lost. Probably many other groups with “unusual” telomeres will be found. We question whether telomerase is indeed the original end-maintenance system and point to other candidate processes involving t-loops, t-circles, rolling circle replication and recombination. Possible evolutionary outcomes arising from the loss of telomerase activity in alternative lengthening of telomere (ALT) systems are discussed. We propose that elongation of minisatellite repeats using recombination/replication processes initially substitutes for the loss of telomerase function. Then in more established ALT groups, subtelomeric satellite repeats may replace the telomeric minisatellite repeat whilst maintaining the recombination/replication mechanisms for telomere elongation. Thereafter a retrotransposition-based end-maintenance system may become established. The influence of changing sequence motifs on the properties of the telomere cap is discussed. The DNA and protein components of telomeres should be regarded – as with any other chromosome elements – as evolving and co-evolving over time and responding to changes in the genome and to environmental stresses. We describe how telomere dysfunction, resulting in end-to-end chromosome fusions, can have a profound effect on chromosome evolution and perhaps even speciation.

Telomere variability in the monocotyledonous plant order Asparagales

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the ‘typical’ Arabidopsis–type telomere (TTTAGGG)n. This stimulated us to determine what has replaced these sequences. Using slot–blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis–type telomeric sequence has been partly or fully replaced by the human–type telomeric sequence (TTAGGG)n. Species in Allium lack the human–type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis, Bombyx (TTAGG)n and Tetrahymena (TTGGGGn. FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human–type motif. These motifs probably form the ‘true telomere’ but the error rate of motif synthesis is higher compared with ‘typical’ plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis–like telomeres to a low–fidelity synthesis of human–like telomeres.

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.

Telomerase activity in plant cells

Telomerase is a ribonucleoprotein enzyme which elongates the G‐rich strand of telomeric DNA to compensate for the progressive reduction in its length due to incomplete replication of chromosome ends, which in human somatic cells leads to cell cycle arrest upon shortening of telomeres to a critical length. To examine the possible involvement of telomerase in metabolism of plant genetic material, we used cells of Nicotiana tabacum strain TBY‐2, a stable long‐term culture which has kept a constant pattern of restriction fragments from chromosome termini during its 6 month period of cultivation in our laboratory. In a direct assay for telomerase, a 5′ end‐labeled plant telomeric oligonucleotide 5′(TTTAGGG)6 3′ was elongated in a TBY‐2 cell extract, showing a pausing pattern which is a characteristic feature of telomerases from other organisms. The elongation was inhibited by RNase A pretreatment of the extract. We conclude that plant cells possess telomerase which is used for maintenance of their telomeres.

Dysfunction of Chromatin Assembly Factor 1 Induces Shortening of Telomeres and Loss of 45S rDNA in Arabidopsis thaliana

Replication-dependent chromatin assembly contributes to the stability of the copy number of the telomeric and 45S rDNA repeats. While only 10 to 20% of the 45S rDNA repeats remain intact in the 5th generation of the CAF1 mutants, transcription is unaltered. The progressive loss of telomeres and 45S rDNA is accompanied by accumulation of anaphase bridges and increased sterility of the plants. Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA.

Interactions of putative telomere-binding proteins in Arabidopsis thaliana: identification of functional TRF2 homolog in plants

Telomere‐binding proteins are required for forming the functional structure of chromosome ends and regulating telomerase action. Although a number of candidate proteins have been identified by homology searches to plant genome databases and tested for their affinity to telomeric DNA sequences in vitro, there are minimal data relevant to their telomeric function. To address this problem, we made a collection of cDNAs of putative telomere‐binding proteins of Arabidopsis thaliana to analyse their protein–protein interactions with the yeast two‐hybrid system. Our results show that one myb‐like protein, AtTRP1, interacts specifically with AtKu70, the latter protein having a previously described role in plant telomere metabolism. In analogy to the interaction between human Ku70 and TRF2 proteins, our results suggest that AtTRP1 is a likely homolog of TRF2. The AtTRP1 domain responsible for AtKu70 interaction occurs between amino acid sequence positions 80 and 269. The protein AtTRB1, a member of the single myb histone (Smh) family, shows self‐interaction and interactions to the Smh family proteins AtTRB2 and AtTRB3. Protein AtTRB1 also interacts with AtPot1, the Arabidopsis homolog of oligonucleotide‐binding‐fold‐containing proteins which bind G‐rich telomeric DNA. In humans, the TRF1‐complex recruits hPot1 to telomeres by protein–protein interactions where it is involved in telomere length regulation. Possibly, AtTRB1 has a similar role in recruiting AtPot1.

Characterization of a new family of tobacco highly repetitive DNA, GRS, specific for the Nicotiana tomentosiformis genomic component

Members of a new family of highly repetitive DNA sequences called GRS were isolated fromNicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180–182 bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (104 copies per haploid genome) and could be derived from eitherNicotiana tomentosiformis orNicotiana otophora, two possible ancestors of the T genome of the amphidiploidN. tabacum. Sequence homology between the HRS60 (Koukalováet al. 1989) and the GRS family has been estimated to be 57%.In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploidN. tabacum. GRS sequences, like HRS60 (Fajkuset al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conservedMbol site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.

Structure—function relationships in telomerase genes

The TERT (telomerase reverse transcriptase) subunit of telomerase is an intensively studied macromolecule due to its key importance in maintaining genome integrity and role in cellular aging and cancer. In an effort to provide an up‐to‐date overview of the topic, we discuss the structure of TERT genes, their alternative splicing products and their functions. Nucleotide databases contain more than 90 full‐length cDNA sequences of telomerase protein subunits. Numerous in silico, in vitro and in vivo experimental techniques have revealed a great deal of structural and functional data describing particular features of the telomerase subunit in various model organisms. We explore whether particular findings are generally applicable to telomerases or species‐specific. We also discuss in an evolutionary context the role of identified functional TERT subdomains.

The telomeric sequence is directly attached to the HRS60 subtelomeric tandem repeat in tobacco chromosomes.

PCR and primers derived from the telomeric repeat (CCCTAAA)n and from the tobacco subtelomeric tandemly repetitive sequence HRS60 (EMBL X12489) were used to amplify the region linking the two loci. A 131 bp PCR product was obtained both from total tobacco DNA and from the DNA fraction enriched for telomeres. Its sequence only consists of the telomeric primer and the attached region of the HRS60 repetitive unit up to the end of the sequence complementary to the HRS60 primer. The site of direct continuity between the two sequences is formed by a (dA)7 tract.