A family of telomere-associated autonomously replicating sequences and their functions in targeted recombination in Hansenula polymorpha DL-1

A family of multiple autonomously replicating sequences (ARSs) which are lo cated at several chromosomal ends of Hansenula polymorpha DL-1 has been ide ntified and characterized, Genomic Southern blotting with an ARS, HARS36, o riginating from the end of a chromosome, as a probe showed several homologu es in the genome of H. polymorpha. Nucleotide sequences of the three fragme nts obtained by a selective cloning for chromosomal ends were nearly identi cal to that of HARS36, All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5'-GGGTGGCG-3' which resemble the te lomeric repeat sequence in other eukaryotes, Transformation of H. polymorph a with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivit y to exonuclease Bal31 digestion of the common DNA fragment in all integran ts confirmed the telomeric origin of HARS36 homologues, suggesting that sev eral chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36, Even though the frequenc ies of targeted recombination were varied among the ends of the chromosomes , the overall frequency was over 96%, The results suggested that the integr ation of the plasmids containing telemeric ARSs occurred largely through ho mologous recombination at the telomeric repeats, which serve as high-freque ncy recombination targets.