Microbiology (1994), 140, 551-557 Printed in Great Britain Characterization of telomeric regions from Ustilago ma ydis Plinio A. Guzmdn and J. Genaro SAnchez Author for correspondence : Plinio Guzman. Tel: + 52 462 5 16 00 ext. 444,447. Fax: + 52 462 5 12 82. Departamentode lngenierla de ‘Iantasf de lnvestigacidn y de Estudios Avanzados del IPN, Unidad Irapuato, Apartado Postal 629, 36500 Irapuato, Gto., Mexico The isolation of telomeres from the phytopathogenic fungus Ustilago maydis is described. The telomeric repeat from the plant Arabidopsis thaliana, ‘ITTAGGG, cross-hybridizes to Bal31 -sensitive fragments of U. maydis DNA and detects many or all of the U. maydis chromosomes separated by pulsed-field gel electrophoresis (PFGE). This telomeric repeat was used to screen a library enriched for chromosome ends. Three clones were isolated which contained the tandemly repeated sequence TTAGGG. This sequence is identical to some known telomere repeats found in humans and other vertebrates as well as in some protozoa and moulds. In addition, the three telomeric clones had an almost identical 376 bp segment of middle-repeated telomere-associated sequences adjacent to the telomeric repeat. This segment hybridized to many or all U. maydis chromosomes separated by PFGE and showed a hybridization pattern in genomic digestions similar to that of the telomeric repeat. These results indicate that in U. maydis the same segment of telomere-associated sequences is located adjacent to the telomeric repeat in many or all chromosomes, which suggests that it may have a common role in chromosome function. Keywords : Ustilago maydis, telomere, chromosome function INTRODUCTION In the last few years a dramatic increase of information has been generated in the molecular analysis of telomeres. These specialized DNA-protein complexes located at the chromosomal termini are required to stabilize the chromo- somes and for the complete replication of DNA at the chromosome end. The telomere consists of tandemly repeated copies of a DNA sequence which contains clusters of G residues oriented 5’ to 3’ towards the chromosome end. In telomeres of Arabidopsis, human, Tetrabmena and Cblamydomonas, among other eukaryotes, this sequence is perfectly repeated with little or no divergence. In others, like Saccbaromyces cerevisiae and Dictyostelitrm, variations of a primary pattern are repeated. A repeated sequence is characteristic for each species and it is assumed to be present in all the chromosomes (Blackburn, 1991 ; Kipling & Cooke, 1992; Zakian, 1989). An identical telomere repeat unit can be present in divergent species, ranging from protozoa to plants and humans. Also, telomeres from one species can stabilize . . . . . . . . . . . . ..... . . . . . , ..... . . . . . . . . . . . , . . . , ... . . . . . . . .... . .. . . . . . .. . . . .. . . . .. . . , . , . , , . , . . .. . . . .. . . . ,,, . ,,, . , . , , . . . , . . ,,,,,,,, . , . ,, . . . . . . ,, . ,,,, . . . , Abbreviations: ARS, autonomously replicating sequence; PFGE, pulsed- field gel electrophoresis; UTAS, Ustilago telomere-associatedsequences. The GenBank accession numbers for the sequences reported in this paper are X77242 and X77243. linear molecules in another one, even if their telomeric sequences are different (Zakian, 1989). The length of this repeated unit varies between species ; in humans, differ- ences in length have been observed depending on the cell type or on donor age (Harley et al., 1990; Hastie et al., 1990). In ciliated protozoa, synthesis of the G-rich strand is catalysed by an enzyme activity named telomere terminal transferase or telomerase. Telomerases are ribo- nucleoprotein enzymes, in which the RNA component contains sequences complementary to the telomeric repeat for which it is the template (Blackburn, 1992). Mutations in a gene that is thought to code for a component of the telomerase in S. cerevisiae, or mutations on the telomerase RNA gene of Tetrabymena, can lead to dramatic pheno- types : telomere shortening, chromosomal instability and cell death (Lumblad & Szostak, 1989; Yu et al., 1990). Studies in S. cerevisiae have determined that the only DNA sequence that is required for telomere function is the simple telomeric repeat. Although middle-repetitive DNA sequences are also often found adjacent to the telomere repeat, these sequences are not required for telomere function. For these sequences, called telomere- associated sequences, roles in recombination, replication and chromosome healing have been suggested; in S. cerevisiae these sequences are highly recombinogenic and 0001-8507 0 1994 SGM 551