DOUBLE-STRANDED CLEAVAGE OF T-DNA AND GENERATION OF SINGLE-STRANDED T-DNA MOLECULES IN ESCHERICHIA-COLI BY A VIRD-ENCODED BORDER-SPECIFIC ENDONUCLEASE FROM AGROBACTERIUM-TUMEFACIENS
DOUBLE-STRANDED CLEAVAGE OF T-DNA AND GENERATION OF SINGLE-STRANDED T-DNA MOLECULES IN ESCHERICHIA-COLI BY A VIRD-ENCODED BORDER-SPECIFIC ENDONUCLEASE FROM AGROBACTERIUM-TUMEFACIENS
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DOI：
10.1128/jb.169.11.5035-5045.1987
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发表时间：
1987-11-01
影响因子：
3.2
通讯作者：
SLIGHTOM, JL
中科院分区：
文献类型：
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作者：
JAYASWAL, RK;VELUTHAMBI, K;SLIGHTOM, JL
对根癌农杆菌Ti质粒pTiA6的病毒D位点进行了测序。对该序列的计算机分析表明，该基因座内有5个可能的开放阅读框架(ORF)。在该基因的远端还发现了另外两个ORF。然而，在体内和体外转录-翻译耦合系统中，只检测到两个表观分子质量分别为16和56千道尔顿的多肽，即ORF1和ORF2的产物。在tac启动子的控制下，将病毒D基因克隆到表达载体pKK223.3中，并将其导入含有微型钛质粒pAL1050的大肠杆菌中。当用异丙基-β-D-硫代半乳糖苷诱导时，病毒D基因产物显示出双链T-DNA边界特异性内切酶活性。缺失分析表明，该酶活性编码在病毒D基因5‘端近1.7kb碱基部分，携带ORF1和ORF2的大部分。ORF1和ORF2都没有单独显示内切酶活性；互补研究表明ORF1和ORF2的产物一起具有这种活性。这个1.7kb碱基对的病毒D位点区域的表达导致T-DNA在边界或附近的双链切割，并在大肠杆菌中产生频率大致相同的单链T-DNA分子。
The virD locus of Agrobacterium tumefaciens Ti plasmid pTiA6 was sequenced. Computer analysis of the sequence indicated five possible open reading frames (ORFs) within this locus. Two additional ORFs were identified distal to this locus. However, only two polypeptides of apparent molecular masses 16 and 56 kilodaltons, the products of ORFs 1 and 2, were detected in Escherichia coli, both in vivo and in an in vitro coupled transcription-translation system. The virD locus was cloned in expression vector pKK223.3 under control of a tac promoter and introduced into an E. coli strain harboring mini-Ti plasmid pAL1050. When induced with isopropyl-.beta.-D-thiogalactopyranoside, the virD gene products exhibited double-stranded T-DNA border-specific endonuclease activity. Deletion analysis demonstrated that this activity is encoded within the 5''-proximal 1.7-kilobase-pair portion of the virD locus that carries ORF 1 and most of ORF 2. Neither ORF 1 nor ORF 2 independently showed endonuclease activity; complementation studies indicated that the products of ORFs 1 and 2 together have this activity. The expression of this 1.7-kilobase-pair region of the virD locus caused double-stranded cleavage of the T-DNA at or near the borders and generated single-stranded T-DNA molecules with approximately equal frequencies in E. coli.