INSERTION SITE-SPECIFICITY OF THE TRANSPOSON TN3

被引:15
作者
DAVIES, CJ [1 ]
HUTCHISON, CA [1 ]
机构
[1] UNIV N CAROLINA, DEPT MICROBIOL & IMMUNOL, CHAPEL HILL, NC 27599 USA
关键词
D O I
10.1093/nar/23.3.507
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
The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpositional 'warm' spots were not a limiting factor but a 935 bp 'cold' spot was completed using a synthetic oligonucleotide primer. Two hundred and twenty three miniTn3 insertion sites from three sequencing projects were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this consensus, so insertion occurs promiscuously (similar to 37% of sites are potential targets). In our sequencing projects, multiply targeted sites always closely matched the consensus, although not all close matches were targeted frequently The 935 bp cold spot showed no unusual features when analysed with the consensus sequence. The consensus can be used to accurately predict likely insertion sites in a new sequence. Synthetic oligonucleotides based on the consensus and a known hot spot for Tn3 were mutagenised. These sequences were not hot spots in our vectors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor such as DNA secondary structure, also plays an important role in target site selection for the transposon Tn3.
引用
收藏
页码:507 / 514
页数:8
相关论文
共 21 条
[1]   A RAPID PROCEDURE FOR DNA SEQUENCING USING TRANSPOSON-PROMOTED DELETIONS IN ESCHERICHIA-COLI [J].
AHMED, A .
GENE, 1985, 39 (2-3) :305-310
[2]  
BANKIER AT, 1987, METHOD ENZYMOL, V155, P51
[3]   CLONING OF CHROMOSOME-I DNA FROM SACCHAROMYCES-CEREVISIAE - ANALYSIS OF THE FUN52-GENE, WHOSE PRODUCT HAS HOMOLOGY TO PROTEIN-KINASES [J].
BARTON, AB ;
DAVIES, CJ ;
HUTCHISON, CA ;
KABACK, DB .
GENE, 1992, 117 (01) :137-140
[4]   TN10 INSERTION SPECIFICITY IS STRONGLY DEPENDENT UPON SEQUENCES IMMEDIATELY ADJACENT TO THE TARGET-SITE CONSENSUS SEQUENCE [J].
BENDER, J ;
KLECKNER, N .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (17) :7996-8000
[5]  
Berg DE, 1989, MOBILE DNA
[6]   STUDIES OF THE SPECIFICITY AND CONTROL OF TRANSPOSITION OF THE TN3 ELEMENT [J].
COHEN, SN ;
CASADABAN, MJ ;
CHOU, J ;
TU, CPD .
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, 1979, 43 :1247-1255
[7]   TN7 - A TARGET SITE-SPECIFIC TRANSPOSON [J].
CRAIG, NL .
MOLECULAR MICROBIOLOGY, 1991, 5 (11) :2569-2573
[8]   A DIRECTED DNA SEQUENCING STRATEGY BASED UPON TN3 TRANSPOSON MUTAGENESIS - APPLICATION TO THE ADE1 LOCUS ON SACCHAROMYCES-CEREVISIAE CHROMOSOME-I [J].
DAVIES, CJ ;
HUTCHISON, CA .
NUCLEIC ACIDS RESEARCH, 1991, 19 (20) :5731-5738
[9]   EFFECTS OF RNA SECONDARY STRUCTURE ON ALTERNATIVE SPLICING OF PRE-MESSENGER RNA - IS FOLDING LIMITED TO A REGION BEHIND THE TRANSCRIBING RNA-POLYMERASE [J].
EPERON, LP ;
GRAHAM, IR ;
GRIFFITHS, AD ;
EPERON, IC .
CELL, 1988, 54 (03) :393-401