Molecular and General Genetics MGG

, Volume 249, Issue 4, pp 447–455

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

  • Moriya Ohkuma
  • Keisuke Kobayashi
  • Shinya Kawai
  • Chel Won Hwang
  • Akinori Ohta
  • Masamich Takagi
Original Paper

Abstract

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.

Key words

Centromere Dicentric plasmid Overexpression Candida maltosa 

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Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Moriya Ohkuma
    • 1
  • Keisuke Kobayashi
    • 1
  • Shinya Kawai
    • 1
  • Chel Won Hwang
    • 1
  • Akinori Ohta
    • 1
  • Masamich Takagi
    • 1
  1. 1.Laboratory of Cellular Genetics, Department of Agricultural ChemistryThe University of TokyoBunkyo-ku, TokyoJapan
  2. 2.The Institute of Physical and Chemical Research (RIKEN)Wako-shi, SaitamaJapan
  3. 3.Department of Applied BioscienceTokyo University of Agriculture and TechnologyFuchu, TokyoJapan
  4. 4.Department of Environmental EngineeringHandong UniversityPohang-KyeongbukKorea

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