ANALYZING EXPRESSION OF THE CALMODULIN AND UBIQUITIN-FUSION GENES OF TRYPANOSOMA-CRUZI USING SIMULTANEOUS, INDEPENDENT DUAL GENE REPLACEMENTS

被引:12
作者
CHUNG, SH [1 ]
GILLESPIE, RD [1 ]
SWINDLE, J [1 ]
机构
[1] UNIV TENNESSEE, DEPT MICROBIOL & IMMUNOL, MEMPHIS, TN 38163 USA
关键词
TRYPANOSOMA CRUZI; TRANSFORMATION; GENE REPLACEMENT; TRANSSPLICING;
D O I
10.1016/0166-6851(94)90012-4
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 describe here a strategy for introducing simultaneous, independent gene replacements into the Trypanosoma cruzi chromosome. The goal of this study was to use two linear DNA fragments to simultaneously replace the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomycin resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. One clone (D6), of thirty G418-resistant clones analyzed, carried the desired dual gene replacement. CDNA sequence analysis indicated that the CAT mRNA was accurately trans-spliced using the previously identified FUS1 mini-exon addition site. However, DNA sequence analysis of the intergenic sequence immediately upstream of the neo(r) gene in clone D6 identified a mutation which altered the pattern of trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.
引用
收藏
页码:95 / 107
页数:13
相关论文
共 25 条
[1]   TRANSSPLICING OF NUCLEAR PRE-MESSENGER-RNAS [J].
AGABIAN, N .
CELL, 1990, 61 (07) :1157-1160
[2]   THE CALMODULIN-UBIQUITIN ASSOCIATED GENES OF TRYPANOSOMA-CRUZI - THEIR IDENTIFICATION AND TRANSCRIPTION [J].
AJIOKA, J ;
SWINDLE, J .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1993, 57 (01) :127-136
[3]   STABLE TRANSFORMATION OF LEPTOMONAS-SEYMOURI BY CIRCULAR EXTRACHROMOSOMAL ELEMENTS [J].
BELLOFATTO, V ;
TORRESMUNOZ, JE ;
CROSS, GAM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (15) :6711-6715
[4]  
CamargO E. P., 1964, Revista do Instituto de Medicina Tropical de Sao Paulo, V6, P93
[5]   LINKAGE OF THE CALMODULIN AND UBIQUITIN LOCI IN TRYPANOSOMA-CRUZI [J].
CHUNG, SH ;
SWINDLE, J .
NUCLEIC ACIDS RESEARCH, 1990, 18 (15) :4561-4569
[6]   USING SIMULTANEOUS, TANDEM GENE REPLACEMENTS TO STUDY EXPRESSION OF THE MULTICOPY UBIQUITIN-FUSION (FUS) GENE FAMILY OF TRYPANOSOMA-CRUZI [J].
GILLESPIE, RD ;
AJIOKA, J ;
SWINDLE, J .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1993, 60 (02) :281-292
[7]   CONSERVED SEQUENCES AND TRANSCRIPTION OF THE HSP70-GENE FAMILY IN TRYPANOSOMA-BRUCEI [J].
GLASS, DJ ;
POLVERE, RI ;
VANDERPLOEG, LHT .
MOLECULAR AND CELLULAR BIOLOGY, 1986, 6 (12) :4657-4666
[8]   APPARENT GENERATION OF A SEGMENTED MESSENGER-RNA FROM 2 SEPARATE TANDEM GENE FAMILIES IN TRYPANOSOMA-CRUZI [J].
GONZALEZ, A ;
LERNER, TJ ;
HUECAS, M ;
SOSAPINEDA, B ;
NOGUEIRA, N ;
LIZARDI, PM .
NUCLEIC ACIDS RESEARCH, 1985, 13 (16) :5789-5804
[9]   STABLE TRANSFORMATION OF TRYPANOSOMA-CRUZI - INACTIVATION OF THE PUB12.5-POLYUBIQUITIN GENE BY TARGETED GENE DISRUPTION [J].
HARIHARAN, S ;
AJIOKA, J ;
SWINDLE, J .
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 1993, 57 (01) :15-30
[10]   MATURATION OF POLYCISTRONIC PRE-MESSENGER-RNA IN TRYPANOSOMA-BRUCEI - ANALYSIS OF TRANS SPLICING AND POLY(A) ADDITION AT NASCENT RNA TRANSCRIPTS FROM THE HSP70 LOCUS [J].
HUANG, J ;
VANDERPLOEG, LHT .
MOLECULAR AND CELLULAR BIOLOGY, 1991, 11 (06) :3180-3190