EXCISIVE RECOMBINATION OF THE SLP1 ELEMENT IN STREPTOMYCES-LIVIDANS IS MEDIATED BY INT AND ENHANCED BY XIS
EXCISIVE RECOMBINATION OF THE SLP1 ELEMENT IN STREPTOMYCES-LIVIDANS IS MEDIATED BY INT AND ENHANCED BY XIS
复制标题
DOI：
10.1128/jb.175.10.3075-3082.1993
复制
发表时间：
1993-05-01
影响因子：
3.2
通讯作者：
COHEN, SN
中科院分区：
文献类型：
--
作者：
BRASCH, MA;COHEN, SN
介导SLP1元件定点重组的功能已被定位到一个2.2kb的区域，该区域包括整合位点(AttP)、指定SLP1整合所必需和充分的功能的基因(Int)以及怀疑编码蛋白质Xis的开放阅读框架orf61，该框架与其他位点特异性重组系统的切除酶显示出有限的相似性。在这里，我们描述了研究orf61和int在SLP1切除中各自作用的实验。我们构建了从转录融合到Tn5 aph基因的高拷贝数链霉菌表达orf61、int或同时表达orf61和int的pIJ101的衍生物，并测试了这些构建物促进含有attP的不定质粒的切除能力，该不定质粒已被特异性地整合到变铅青链霉菌染色体的attB位点。仅表达来自外源启动子的INT基因产物就足以切除整合的质粒。这一结果表明SLP1整合型编码蛋白既可以进行激活型重组，也可以进行整合型重组。当orf61基因产物从外源启动子表达时，抑制了染色体attB位点的int介导的整合。此外，在正常发生切除和转移的条件下，orf61编码的蛋白提高了SLP1的精确切除。基于这些发现，我们将orf61基因命名为XIS。
The functions mediating site-specific recombination of the SLP1 element have been mapped to a 2.2-kb region that includes the site of integration (attP), a gene (int) that specifies a function both necessary and sufficient for integration of SLP1, and an open reading frame, orf61, suspected of encoding a protein, Xis, that shows limited similarity to the excisionases of other site-specific recombination systems. Here we describe experiments that investigate the respective roles of orf61 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pIJ101 that express orf61, int, or both orf61 and int from transcriptional fusions to the Tn5 aph gene and tested the ability of these constructs to promote excision of an adventitious attP-containing plasmid that had been integrated site-specifically into the attB site of the Streptomyces lividans chromosome. Expression of the int gene product alone from an exogenous promoter was sufficient for excision of the integrated plasmid. This result indicates that the SLP1 int-encoded protein can carry out excisive, as well as integrative, recombination. The orf61 gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal attB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the orf61-encoded protein. On the basis of these findings, we here designate the orf61 gene as xis.