Structure Analysis Of Plant Telomeres

Telomere is the terminal part of eukaryotic chromosome and can serve as a protective cap for stabilization of chromosomes and prevention of chromosomal end fusions. Telomere may be also associated with the organism aging and tumorigenesis. The telomere structure shows conservation between organisms, which constitutes the tandem repetitive telomeric region, the subtelomeric region, and the linkage region between them. Telomeric chromatin shows a specific feature, compared with other types of chromatin, which has unique chromatin modifications and specific packaging of nucleosomes. In this research, we study the structure of plant telomeres, using maize and Chrysanthemum segetum L as materials. The results are as follows:Subtelomeric sequence is an important part of the telomere structure and different PCR strategies were used to isolate subtelomeric sequences. In this study, a simple method was developed to isolate the subtelomeric sequence in maize, in which two complementary telomeric primers were used respectively in the single primer amplification of genomic DNA for isolating the subtelomeric sequence. A specific amplification band was obtained with telomere C-strand primer. A smear amplification pattern was obtained with telomere G-strand primer. The specific amplified band was cloned and sequenced. A 412 bp clone (designated as MTAS1) showed a high homology with a reported 382 bp maize subtelomere sequence, suggesting that MTASl is a subtelomeric sequence. The MTAS1 showed a novel sequence structure feature that it was flanked by a forward telomeric sequence and an inverted telomere sequence at the two ends. A ladder pattern of MTAS1 signals was seen in the Southern blot hybridization, which demonstrated that MTAS1 was a tandemly repetitive sequence. Fluorescence in situ hybridization (FISH) results revealed that MTAS1 was mainly distributed at the ends of the short arms of some long chromosomes, which confirmed that MTAS1 was a subtelomeric sequence. The brightness of MTAS1 signals suggested that this sequence was a highly repetitive sequence in maize. Dual-FISH with telomere and MTAS1 probes on the chromosomes and genomic fibers revealed that MTAS1 signals were located adjacent to or overlapped with telomere signals and distributed heterogeneously in subtelomeric regions of several chromosomes, even at two ends of a single chromosome.Several reports revealed that the nucleolar organizer regions (NORs) were located at the telomeric regions of chromosomes and exhibited a close structure connection at cellular level with telomere in many organisms. In Chrysanthemum segetum L., we found by accident that all the 45S rDNAs were located at the telomeric regions of chromosomes. The rDNA showed different signal patterns among the observed chromosome metaphases. Fiber-FISH with rDNA and telomere probes revealed that the telomere signals were closely connected with or interspersed into rDNA signals on the fibers. Based on these cytogical observations, we assumed that specific sequences flanked by rDNA and telomere sequences might be obtained using a combination of telomere and rDNA primers. One representative clone designated as CHS2 showed a close connection between rDNA and telomere sequences, which suggested that the telomere sequence was inserted into the conservative rDNA sequence. Seveal other clones were found to be flanked by the single telomere primer or rDNA primer. Our data implied that homologous recombinations occurred at the chromosomal terminals between the tandem repeated rDNA sequences or telomeric tandem repeats.In vertebrates, some studies revealed that telomeric chromatin similar to percentric heterochromatin was transcribed and generated telomeric repeat-containing RNA (abbreviation:TERRA or TelRNA). TERRA is considered to be an important component of telomeric chromatin and plays a unique role in epigenetically regulating the telomeric chromatin structure and telomerase activity. In this study, we investigated differential expression of telomeric repeat-containing RNA (TERRA) in different maize tissues. Reverse transcription polymerase chain reactions (RT-PCR) with telomere primers could not detect the TERRA, so the subtelomeric sequence primers close to the telomere were employed to detect the transcription of telomeric chromatin. The subtelomeric primers were designed from the maize subtelomeric sequence MTAS1. The product of RT-PCR was cloned and sequenced. These clones contained both a part of a telomere homologous sequence and a part of a subtelomere homologous sequence, which confirmed that the telomeric chromatin was transcribed and the TERRA existed in maize. The real-time PCR results showed that the expression of MTAS1 was quantitatively specific between tissues with different developmental states, suggesting that the transcription of TERRA was developmentally regulated. In addition, the telomerase activity was detected by a telomeric repeat amplification protocol (TRAP) among those tissues and the results showed that the highest telomerase activity was detected in the tissue with the highest expression level of TERRA, suggesting the possible regulation of telomerase activity by the level of TERRA.