APPLICATION OF THE BETA-GLUCURONIDASE GENE FUSION SYSTEM TO SACCHAROMYCES-CEREVISIAE

被引:36
作者
SCHMITZ, UK
LONSDALE, DM
JEFFERSON, RA
机构
[1] INST PLANT SCI RES,DEPT MOLEC GENET,CAMBRIDGE CB2 2JB,ENGLAND
[2] INT ATOM AGCY,A-1400 VIENNA,AUSTRIA
关键词
Chimeric genes; Protein targeting; Reporter gene; Transformation;
D O I
10.1007/BF00312618
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
Bacterial β-glucuronidase (GUS) has been described as a useful reporter enzyme for gene fusion studies in bacteria and plants. Here we report the expression of GUS in yeast to illustrate further applications of this enzyme as a quantitative tool for measuring gene activity, as a colour selection marker and as a versatile system for protein targeting studies. There is no intrinsic GUS activity in any yeast strain tested. GUS was expressed in transgenic yeast on a multiple-copy vector under the control of the alcohol dehydrogenase 1 (ADH1) promoter. The enzyme is stable in yeast and its activity may be monitored by very sensitive colorimetric or fluorometric methods in extracts, or by the histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X-Gluc) on plates. To test the efficacy of GUS as a reporter for targeting proteins into different subcellular compartments in vivo, we fused the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene (MSW) to the amino terminus of GUS. The activity of the fusion protein is not substantially impaired and it is imported efficiently into yeast mitochondria. © 1990 Springer-Verlag.
引用
收藏
页码:261 / 264
页数:4
相关论文
共 19 条
[1]  
AMMERER G, 1983, METHOD ENZYMOL, V101, P192
[2]  
BENNETZEN JL, 1982, J BIOL CHEM, V257, P3018
[3]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[4]   AN ELECTROPHORETIC KARYOTYPE FOR YEAST [J].
CARLE, GF ;
OLSON, MV .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (11) :3756-3760
[5]  
DAUM G, 1982, J BIOL CHEM, V257, P3028
[6]   HOW PROTEINS FIND MITOCHONDRIA AND INTRAMITOCHONDRIAL COMPARTMENTS [J].
HURT, EC ;
VANLOON, APGM .
TRENDS IN BIOCHEMICAL SCIENCES, 1986, 11 (05) :204-207
[7]   TRANSFORMATION OF INTACT YEAST-CELLS TREATED WITH ALKALI CATIONS [J].
ITO, H ;
FUKUDA, Y ;
MURATA, K ;
KIMURA, A .
JOURNAL OF BACTERIOLOGY, 1983, 153 (01) :163-168
[8]  
Jefferson R.A., 1987, PLANT MOL BIOL REP, V5, P387, DOI DOI 10.1007/BF02667740
[9]  
JEFFERSON RA, 1987, EMBO J, V6, P3901
[10]   BETA-GLUCURONIDASE FROM ESCHERICHIA-COLI AS A GENE-FUSION MARKER [J].
JEFFERSON, RA ;
BURGESS, SM ;
HIRSH, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (22) :8447-8451