A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in Hansenula polymorpha DL-1

Abstract

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome ofH. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical 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 telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha 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 sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.

Inactivation of theHansenula polymorpha PMR1gene affects cell viability and functioning of the secretory pathway

“…The plasmid pBB deriving from pE1 was constructed by the replacement of the BamHI-BstEII fragment inside the PMR1 ORF with the SnaBI-BamHI fragment of pCHLX carrying the LEU2 marker (Sohn et al, 1996). The plasmid pAF14 was constructed by the replacement of the StuI-NheI fragment in the pE1 plasmid with the HincII-NheI fragment of the pGAG418-43 plasmid carrying G418 R (Sohn et al, 1999). The plasmid pCAT1 was constructed by the insertion of the SmaI-Psp124BI fragment of the pE1 plasmid carrying the HpPMR1 gene between the HpaI and Psp124BI sites of the p2CHA6 plasmid possessing the HpADE2 marker (M. Agaphonov, unpublished).…”

Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast

“…The function of the subtelomeric sequence and the reason why overexpression of X-element causes growth inhibition have not yet been revealed. The presence of ARS consensus at the subtelomeric regions was also reported in Hansenula polymorpha (Sohn et al, 1999), suggesting that the subtelomeric regions played an important role for chromosomal replication at the ends.…”

Genomic Exploration of the Hemiascomycetous Yeasts: 13. Pichia angusta

“…All RSTs were, nevertheless, compared to the complete sequence of the S. cerevisiae 2‐micron plasmid but no similarity was found. In addition, no RST was similar to the autonomously replicating sequence HARS36 of the P. angusta DL‐1 strain [20].…”