A tetracycline-inducible gene expression system in Entamoeba histolytica

被引:62
作者
Ramakrishnan, G
Vines, RR
Mann, BJ
Petri, WA
机构
[1] UNIV VIRGINIA,HLTH SCI CTR,DEPT MED,CHARLOTTESVILLE,VA 22908
[2] UNIV VIRGINIA,HLTH SCI CTR,DEPT MICROBIOL,CHARLOTTESVILLE,VA 22908
[3] UNIV VIRGINIA,HLTH SCI CTR,DEPT PATHOL,CHARLOTTESVILLE,VA 22908
关键词
Entamoeba histolytica; inducible promoter; tetracycline; hygromycin; tetR;
D O I
10.1016/S0166-6851(96)02784-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have developed an episomal inducible gene expression system in Entamoeba histolytica based on the TetR repressor. The tetR gene was placed under control of 5' and 3' ferredoxin (fdx) regulatory sequences on a plasmid encoding the hygromycin resistance gene directed by 5' and 3' hgl sequences. The reporter luciferase constructs were introduced on a second episome bearing the neomycin resistance gene controlled by 5' and 3' actin sequences. The reporter constructs were driven by the hgl5 promoter in which the tetO sequence was introduced. We found that the optimal tetO location for induction by tetracycline was +4 from the start of transcription. The efficiency of repression and the induction ratio could be improved by increasing hygromycin levels, presumably by increasing tetR plasmid levels. Under these conditions, maximal induction of reporter luciferase could be effected with 5 mu g/ml tetracycline in 18 h. This system permits regulated expression of the reporter gene over two orders of magnitude and should be useful in the analysis of gene function. Copyright (C) 1997 Elsevier Science B.V.
引用
收藏
页码:93 / 100
页数:8
相关论文
共 22 条
[1]   UNUSUAL GENE ORGANIZATION IN THE PROTOZOAN PARASITE ENTAMOEBA-HISTOLYTICA [J].
BRUCHHAUS, I ;
LEIPPE, M ;
LIOUTAS, C ;
TANNICH, E .
DNA AND CELL BIOLOGY, 1993, 12 (10) :925-933
[2]   ANALYSIS OF THE 170-KDA LECTIN GENE PROMOTER OF ENTAMOEBA-HISTOLYTICA [J].
BUSS, H ;
LIOUTAS, C ;
DOBINSKY, S ;
NICKEL, R ;
TANNICH, E .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1995, 72 (1-2) :1-10
[3]   DOUBLE TARGETED GENE REPLACEMENT FOR CREATING NULL MUTANTS [J].
CRUZ, A ;
COBURN, CM ;
BEVERLEY, SM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (16) :7170-7174
[4]   NEW MEDIUM FOR AXENIC CULTIVATION OF ENTAMOEBA-HISTOLYTICA AND OTHER ENTAMOEBA [J].
DIAMOND, LS ;
HARLOW, DR ;
CUNNICK, CC .
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, 1978, 72 (04) :431-432
[5]   RNA POLYMERASE-III CATALYZED TRANSCRIPTION CAN BE REGULATED IN SACCHAROMYCES-CEREVISIAE BY THE BACTERIAL TETRACYCLINE REPRESSOR OPERATOR SYSTEM [J].
DINGERMANN, T ;
FRANKSTOLL, U ;
WERNER, H ;
WISSMANN, A ;
HILLEN, W ;
JACQUET, M ;
MARSCHALEK, R .
EMBO JOURNAL, 1992, 11 (04) :1487-1492
[6]   ESTABLISHMENT OF A SYSTEM FOR CONDITIONAL GENE-EXPRESSION USING AN INDUCIBLE TRANSFER-RNA SUPPRESSOR GENE [J].
DINGERMANN, T ;
WERNER, H ;
SCHUTZ, A ;
ZUNDORF, I ;
NERKE, K ;
KNECHT, D ;
MARSCHALEK, R .
MOLECULAR AND CELLULAR BIOLOGY, 1992, 12 (09) :4038-4045
[7]   CONSTRUCTION OF A TETRACYCLINE-INDUCIBLE PROMOTER IN SCHIZOSACCHAROMYCES-POMBE [J].
FARYAR, K ;
GATZ, C .
CURRENT GENETICS, 1992, 21 (4-5) :345-349
[8]   CHARACTERIZATION OF THE INTERACTION OF PLANT TRANSCRIPTION FACTORS USING A BACTERIAL REPRESSOR PROTEIN [J].
FROHBERG, C ;
HEINS, L ;
GATZ, C .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (23) :10470-10474
[9]   TN10-ENCODED TET-REPRESSOR CAN REGULATE AN OPERATOR-CONTAINING PLANT PROMOTER [J].
GATZ, C ;
QUAIL, PH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (05) :1394-1397
[10]   STRINGENT REPRESSION AND HOMOGENEOUS DE-REPRESSION BY TETRACYCLINE OF A MODIFIED CAMV 35S PROMOTER IN INTACT TRANSGENIC TOBACCO PLANTS [J].
GATZ, C ;
FROHBERG, C ;
WENDENBURG, R .
PLANT JOURNAL, 1992, 2 (03) :397-404