35. Isolation and characterization of a telomere-like sequence in rice

Tiyun WU1, Sandra ZHANG1, Jinhua XIAO2, Hsin-Kan WU3 and Ray Wu1

1) Section of Biochemistry, Molecular & Cell Biology, and
2) Department of Plant Breeding and Biometry, Cornell University, Ithaca, New York 14853, U.S.A.;
3) Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan, R.O.C.

A telomere can be functionally defined as a region of DNA at the end of a linear chromosome that is required for replication and stability of the chromosome. Telomeric DNA consists of short repeat units, usually 6-7 bp in length (the repeat unit is called a monomeric unit) that are tandemly repeated from a few up to several hundred times (Zakian 1989). Here we report the identification and characterization of a telomere-like sequence from rice.

To study rice telomere and telomere-like sequences, we isolated several clones from a partial Sau3A-digested rice genomic library by using an Arbidopsis telomeric DNA, pAtT4 (Richards and Ausubel 1988), as a hybridization probe. A 2.5-kb insert from clone #5, which hybridized to the probe pAtT4, was subcloned into pTZ19 vector. This clone (pOsTel-L) was further characterized and a restriction map is shown in Fig. 1A. The 2.5-kb insert contains 11 tandemly-repeated copies of a telomere-like sequence, CCCTAAA, between nucleotides 1703 and 1777. The sequence of a StyI-SpeI fragment which includes the telomere-like sequence is shown in Fig. 1B.

A homology search against the GEN EMBL detabased of 1992 was carried out to analyze the 2.5-kb sequence including the telomere-like repeats. Significant sequence similarity to the OsTel-L sequence was found. The monomeric unit of the rice telomeric repeat is identical to the Arabidopsis telomeric sequence and shows similarity or identity to all of the published telomeric sequences or telomere-associated repetitive sequences from plants and mammals.

Although the telomere-like repeats are located in the middle of the cloned DNA, the presence of a Sau3A site only 5 bp to the repeats suggests that this 2.5-kb DNA fragment may have come from a recombination of two smaller DNA fragments during library construction. To test this possibility, we cleaved the 2.5-kb DNA into three fragments, then used each fragment as a probe for genomic Southern blot and RFLP analyses. Results showed that fragment TW-400 (Fig. 1A) does not contain the telomere-like repeats and it is not linked to the remaining sequence in pOsTel-L (data not shown).

Fig. 1. Physical map and partial nucleotide sequence of pOsTel-L. (A) An abbreviated restriction map of pOsTel-L. Solid bar shows the cluster of 11 copies of the telomeric sequence CCCTAAA; open bar shows regions that flank the telomere-like sequence; striped bar shows an unrelated sequences. (B) DNA sequence of a Sty I-Spe I fragment.

To determine if the telomere-like repeats are indeed coming from the telomeric regions of a chromosome, we performed Southern blot analysis on Bal31 nuclease-treated rice genomic DNA (Zakian 1989). Genomic DNA was first digested with the exonuclease Bal31 for different lengths of time and then cut with Rsa I or Taq I. After electrophoresis, the Southern blot filter was probed with the cloned Arabidopsis telomeric probe. Fig. 2A shows that the size and intensity of the heterodispersed bands decrease drastically upon increasing time of exonuclease digestion and that there are no visible hybridization signals in lanes 4 and 5. Fig. 2B shows the hybridization pattern of the same Bal3l-treated genomic DNA probe with the rice telomeric fragment, pOsTel-L1.4. As can be seen, the major pattern of hybridization in Fig. 2B is the same as that in Fig. 2A. Furthermore, Fig. 2B shows the presence of two distinct bands (around 1.8 kb and 2.4 kb, respectively) on either the Rsa I- or the Taq I-digested BaI31-treated genomic DNA, indicating that the probe also contains non-telomeric sequences which are closely linked to the rice telomeric sequences. To prove that the exonuclease susceptibility of rice genomic DNA sequences that hybridized to pAtT4 and oOsTel-L1.4 sequences was not due to over-digestion of the genomic sequence or as a property of genomic sequences in general, we reprobed the filter shown in Fig. 2A with rice ribosomal DNA. The result (data not shown) indicated that the rRNA gene probe hybridized to several bands, none of which shifted mobility nor decreased in hybridization intensty. Again, the pattern of pOsTel-L1.4 hybridization to the exonuclease-susceptible restriction fragment was the same as that of pAtT4.

Additional evidence to show that OsTel-L1.4 is the sequence which immediately flanks the telomeric sequences was obtained from an RFLP mapping analysis. Results in Fig. 3 show that the DNA fragment, TW-500, was mapped to the very end of rice chromosome 2. Since DNA fragment TW-500 is immediately adjacent to pOsTel-L1.4, which contains the rice telomere-like repeats, the latter fragment must be located at the very end of chromosome 2 as well. Additional confirmation comes from in situ hybridization which shows that a majority of the signal was located in each end of the rice chromosome (see Res. Note 34).

Fig. 2. pAtT4 and pOsTel-L1.4 hybridization to Bal3l-treated rice genomic DNA. (A) Rice (IR36) genomic DNA was treated with 2.5 units of Bal3l for 0 (lane 1), 20 (lane 2), 40 (lane 3), 60 (lane 4) or 80 (lane 5) minutes and subsequently digested with RsaI (left lanes) or TaqI (right lanes). The DNAs were then size-fractionated by electrophoresis through a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with radiolabeled pAtT4 and washed at high stringency. (B) A duplicated membrane shown in (A) was hybridized with radiolabeled pOsTel-L1.4 which contains 11 copies of telomere-like sequences from rice and washed at high stringency.

Fig. 3. Molecular linkage maps of the rice chromosome 2 showing the location of the DNA fragment TW-500. (A) The entire chromosome 2, based on a backcross population derived from the interspecific cross O. sativa X O. longistaminata. (B) A part of chromosome 2 derived from the intersubspecific cross IndicaxJaponica. Both maps were constructed in Dr. Tanksley's laboratory at Cornell University, USA.

References

Richards, E.J. and F.M. Ausubel, 1988. Isolation of higher eukaryotic telomere from Arabidopsis thaliana. Cell 53: 127-136.

Tanksley, S., M. Causse, T. Fulton, N. Ahn, Z. Wang, K. Wu, J. Ziao, Z. Yu, G. Second and S. McCouch, 1991. A high density molecular map of the rice genome. RGN 9: 111-115.

Zakian, V. A., 1989. Structure and function of telomeres. Ann. Rev. Genet. 23: 579-604.