Keggins K M, Lovett P S, Marrero R, Hoch S O
J Bacteriol. 1979 Sep;139(3):1001-6. doi: 10.1128/jb.139.3.1001-1006.1979.
Plasmid pUB110 was previously used as a vector to clone fragments of deoxyribonucleic acid that complement the trpC2 mutation in Bacillus subtilis from endonuclease EcoRI digested B. licheniformis, B. pumilus, and B. subtilis cellular deoxyribonucleic acid. Each of several such trp plasmids was subsequently shown to contain a segment of the trp gene cluster on the basis of genetic complementing activity. In the present study, analysis of the Trp enzyme levels in B. subtilis harboring the constructed trp plasmids confirms the genetic constitution of the plasmids. Thus, plasmids that complement mutations in specific trp genes specify the corresponding enzyme activities. The levels of the plasmid-specified Trp enzymes in B. subtilis were generally above the repressed level of the chromosomally specified Trp enzymes and equal to or below the derepressed levels of the chromosomally specified Trp enzymes. Certain cloned trp segments contain a single HindIII-sensitive site. Insertion of HindIII-generated deoxyribonucleic acid fragments into these trp plasmids resulted in inactivation of trpC complementing activity, loss of the trpC-specified enzyme activity, and a 10-fold reduction in the specific activity of the plasmid-specified trpF product. The HindIII insertions had no detectable effect on the level of the trpD product, nor did the insertions detectably alter plasmid-specified complementing activity other than to abolish trpC complementation. Removal of the HindIII insertions was accompanied by recovery of trpC complementing activity and restoration of the trpC-and trpF-determined enzymes to the levels specified by the parent plasmids.
质粒pUB110先前被用作载体，以克隆来自经核酸内切酶EcoRI消化的地衣芽孢杆菌、短小芽孢杆菌和枯草芽孢杆菌细胞脱氧核糖核酸中能互补枯草芽孢杆菌trpC2突变的脱氧核糖核酸片段。随后，基于遗传互补活性，证明了几个这样的trp质粒中的每一个都含有trp基因簇的一个片段。在本研究中，对携带构建的trp质粒的枯草芽孢杆菌中Trp酶水平的分析证实了这些质粒的遗传组成。因此，能互补特定trp基因突变的质粒可产生相应的酶活性。枯草芽孢杆菌中质粒指定的Trp酶水平通常高于染色体指定的Trp酶的阻遏水平，且等于或低于染色体指定的Trp酶的去阻遏水平。某些克隆的trp片段含有一个单一的HindIII敏感位点。将HindIII产生的脱氧核糖核酸片段插入这些trp质粒导致trpC互补活性失活、trpC指定的酶活性丧失，以及质粒指定的trpF产物的比活性降低10倍。HindIII插入对trpD产物的水平没有可检测到的影响，除了消除trpC互补作用外，插入也未可检测到地改变质粒指定的互补活性。去除HindIII插入片段伴随着trpC互补活性的恢复以及trpC和trpF决定的酶恢复到亲本质粒指定的水平。
