Temporal and spatial control of flagellar and chemotaxis gene expression during Caulobacter cell differentiation.
Temporal and spatial control of flagellar and chemotaxis gene expression during Caulobacter cell differentiation.
复制标题
柄杆菌细胞分化过程中鞭毛和趋化基因表达的时间和空间控制。
DOI：
10.1101/sqb.1985.050.01.101
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Shapiro,L
中科院分区：
文献类型：
--
作者：
Champer,R;Bryan,R;Gomes,SL;Purucker,M;Shapiro,L
方法Tn 5-半乳糖苷酶启动子探针的制备。TnS-RC 96转座子的构建（图2，图I）利用了在新霉素磷酸转移酶II（NPT-II）启动子探针Tn 5-VB 32的构建中首次采用的策略（Bellofatto等，1984）。通过将无启动子的α-半乳糖苷酶基因插入大肠杆菌质粒pRZ 341（ColE 1：：Tn 5 -341）中产生pRC 96（约翰逊和Reznikoff 1983）。编码NPT-II和大部分IS 50 L的基因从pRZ 341中的Tn 5插入物中缺失，并携带来自编码四环素抗性的Tn 10的限制性片段（泰特R）（图2，图I）。将含有trp操纵子的内部序列（在所有三个阅读框中具有翻译终止以防止融合蛋白的形成）的pMC 871的7.1-kb限制性片段（Casadaban等，1980），随后是无启动子的lacZ基因和lacY基因，插入pRZ 341中邻近IS 50 L的剩余部分。为了促进Tn 5-RC 96转移到C. crescentus中，将其插入pJB 9 JI中，pJB 9 JI是一种编码庆大霉素抗性（Gm R）和壮观霉素抗性（Sp R）并含有噬菌体Mu的自传递P型R质粒（Beringer等，1978）。质粒pRC 96
METHODSGeneration of the Tn5~-galactosidase promoter probe. Construction of the transposon of TnS-RC96 (Fig. 2, panel I) utilized a strategy first employed in the construction of the neomycin phosphotransferase II (NPT-II) promoter probe Tn5-VB32 (Bellofatto et al. 1984). pRC96 was generated by inserting a promoterless~-galactosidase gene into the Escherichia coli plasmid pRZ341 (ColEl:: Tn5-341)(Johnson and Reznikoff 1983). The gene encoding NPT-II and most of IS50L is deleted from the Tn5 insert in pRZ341 and carries a restriction fragment from TnlO encoding tetracycline resistance (Tet R)(Fig. 2, panel I). A 7.1-kb restriction fragment of pMC871 (Casadaban et al. 1980) containing internal sequences of the trp operon (with translational stops in all three reading frames to prevent the formation of fusion proteins), followed by a promoterless lacZ gene and a lacY gene, was inserted adjacent to the remaining portion of IS50L in pRZ341. To facilitate transfer of Tn5-RC96 into C. crescentus, it was inserted into pJB9JI, a self-transmissible P-type R plasmid encoding gentamycin resistance (Gm R) and spectinomycin resistance (Sp R) and containing bacteriophage Mu (Beringer et al. 1978). The plasmid pRC96