Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

Abstract:

:For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Adachi N,Ikeda H,Kikuchi A

doi

10.1093/nar/22.20.4229

subject

Has Abstract

pub_date

1994-10-11 00:00:00

pages

4229-33

issue

20

eissn

0305-1048

issn

1362-4962

journal_volume

22

pub_type

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