Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats.
The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG)( n ) was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/μg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura(+) mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan. shoutaka@vos.nagaokaut.ac.jp
Shouji
S
Takahashi
Yumie
Y
Nakajima
Tomomi
T
Imaizumi
Yudai
Y
Furuta
Yuichi
Y
Ohshiro
Katsumasa
K
Abe
Ryo-Hei
RH
Yamada
Yoshio
Y
Kera
D003454:Cryptococcus / Q000235:genetics*; D004261:DNA Replication; D018628:Gene Dosage; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere; D014170:Transformation, Genetic