Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus
Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus
复制标题
嗜热链球菌中的定向基因组整合、基因替换和整合基因表达
DOI：
--
复制
发表时间：
1993
影响因子：
3.2
通讯作者：
MICHkLE DELLEY1
中科院分区：
文献类型：
--
作者：
B. Mollet;J. Knol;B. Poolman;Olivier Marciset;MICHkLE DELLEY1
几个含有红霉素抗性标记的pGEM5和pUC19衍生质粒被整合到嗜热链球菌的染色体上乳糖代谢基因的基因座上。整合通过同源重组发生，并在用于整合的同源DNA的两侧导致了质粒和基因组之间的共整合。对红霉素抗性基因的选择压力导致整合的质粒的多重扩增。然而，这种选择性压力的释放让位于协整结构的相应拆分。通过整合和随后的解析，我们能够用携带体外产生的缺失的修改副本来取代染色体上的LacZ基因。以同样的方法，我们在乳糖操纵子的染色体Lacs和LacZ基因之间整合了一个无启动子的氯霉素乙酰转移酶(CAT)基因。插入的CAT基因成为操纵子的一个功能部分，并相应地表达和调节。在牛奶的正常生长条件下，对必需的Lacs和LacZ基因施加选择性压力，确保了整合基因的维持和表达。由于插入的CAT基因两侧只有很少的重复DNA序列(NdeI位点)，因此即使在没有乳糖的情况下，即当生长在蔗糖或葡萄糖上时，它也能稳定地保持。这种方法代表了一个稳定的系统，可以在嗜热链霉菌中表达和调节外源基因，这种基因在未来可能有资格用于食品。
Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.
使用质粒 pVA891 克隆库进行随机诱变后，从变形链球菌乳糖阴性突变体中克隆大肠杆菌中的磷酸-β-半乳糖苷酶基因。
影响因子：
2.1
作者：
Sato,Y;Yamamoto,Y;Kizaki,H;Kuramitsu,HK
通讯作者：
Kuramitsu,HK
将单拷贝序列引入大肠杆菌染色体：应用于基因和操纵子融合。
影响因子：
3.5
作者：
Resnik,E;LaPorte,DC
通讯作者：
LaPorte,DC
影响因子：
3.5
作者：
MACRINA, FL;TOBIAN, JA;CLEWELL, DB
通讯作者：
CLEWELL, DB