AN IMPROVED VECTOR SYSTEM FOR CONSTRUCTING TRANSCRIPTIONAL LACZ FUSIONS - ANALYSIS OF REGULATION OF THE DNAA, DNAN, RECF AND GYRB GENES OF ESCHERICHIA-COLI

被引:41
作者
MACIAN, F [1 ]
PEREZROGER, I [1 ]
ARMENGOD, ME [1 ]
机构
[1] FDN VALENCIA INVEST BIOMED, INST INVEST CITOL, E-46010 VALENCIA, SPAIN
关键词
RECOMBINANT DNA; GENE FUSIONS; PROMOTER DETECTION; TRANSCRIPTIONAL TERMINATORS;
D O I
10.1016/0378-1119(94)90317-4
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.
引用
收藏
页码:17 / 24
页数:8
相关论文
共 32 条
[1]   DNA-SEQUENCE AND TRANSCRIPTION OF THE REGION UPSTREAM OF THE ESCHERICHIA-COLI GYRB-GENE [J].
ADACHI, T ;
MIZUUCHI, K ;
MENZEL, R ;
GELLERT, M .
NUCLEIC ACIDS RESEARCH, 1984, 12 (16) :6389-6395
[2]   INVIVO ANALYSIS OF THE MECHANISMS RESPONSIBLE FOR STRONG TRANSCRIPTIONAL POLARITY IN A SENSE MUTANT WITHIN AN INTERCISTRONIC REGION [J].
ALIFANO, P ;
CIAMPI, MS ;
NAPPO, AG ;
BRUNI, CB ;
CARLOMAGNO, MS .
CELL, 1988, 55 (02) :351-360
[3]   INTERACTION OF THE BACILLUS-SUBTILIS DNAA-LIKE PROTEIN WITH THE ESCHERICHIA-COLI DNAA PROTEIN [J].
ANDRUP, L ;
ATLUNG, T ;
OGASAWARA, N ;
YOSHIKAWA, H ;
HANSEN, FG .
JOURNAL OF BACTERIOLOGY, 1988, 170 (03) :1333-1338
[4]   OVERLAPPING ARRANGEMENT OF THE RECF AND DNAN OPERONS OF ESCHERICHIA-COLI - POSITIVE AND NEGATIVE CONTROL SEQUENCES [J].
ARMENGOD, ME ;
LAMBIES, E .
GENE, 1986, 43 (03) :183-196
[5]  
ARMENGOD ME, 1988, J BIOL CHEM, V263, P12109
[6]  
ARMENGOD ME, 1991, J BIOL CHEM, V266, P19725
[7]   AUTO-REGULATION OF THE DNAA GENE OF ESCHERICHIA-COLI-K12 [J].
ATLUNG, T ;
CLAUSEN, ES ;
HANSEN, FG .
MOLECULAR & GENERAL GENETICS, 1985, 200 (03) :442-450
[8]   MOLECULAR ANALYSIS OF THE RECF GENE OF ESCHERICHIA-COLI [J].
BLANAR, MA ;
SANDLER, SJ ;
ARMENGOD, ME ;
REAM, LW ;
CLARK, AJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (15) :4622-4626
[9]   AUTO-REGULATION OF THE DNA-REPLICATION GENE DNAA IN ESCHERICHIA-COLI K-12 [J].
BRAUN, RE ;
ODAY, K ;
WRIGHT, A .
CELL, 1985, 40 (01) :159-169
[10]  
BROSIUS J, 1987, METHOD ENZYMOL, V153, P54