Heidecker G, Müller-Hill B
Mol Gen Genet. 1977 Oct 24;155(3):301-7. doi: 10.1007/BF00272809.
Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and beta-galactosidase. The mutants were isolated as TonB- Lac+ derivatives of a phenotypically Lac- TrpR- strain carrying a lac I+ -Z+ fusion on a phi80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA- and 3 Trp- strains produce triple chimeras. The chimeras from the two TrpA- strians were further examined. They consist of tryptophan synthetase alpha-subunit, lac repressor and beta-galactosidase. In crude extracts of these strains the tryptophan synthetase alpha-subunit part can be identified by its ability to aggregate with the beta-subunit since some of the beta-subunit activity can be precipitated with antiserum against beta-galactosidase. Furthermore beta-galactosidase precipitates with antiserum against tryptophan synthetase alpha-subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The beta-galactosidase part is as unaffected as in the original lac repressor-beta-galactosidase chimera. The molecular weights of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.
分离出了几种大肠杆菌突变体，它们在色氨酸酶之一、乳糖阻遏物和β-半乳糖苷酶之间产生三重嵌合体。这些突变体是从一个携带φ80dlac噬菌体上的lac I+-Z+融合基因的表型为Lac-TrpR-的菌株的TonB-Lac+衍生物中分离出来的。噬菌体以这样一种方式整合到染色体中，即lac和trp基因沿相同方向转录。在总共58个候选菌株中，2个TrpA-和3个Trp-菌株产生三重嵌合体。对来自两个TrpA-菌株的嵌合体进行了进一步研究。它们由色氨酸合成酶α亚基、乳糖阻遏物和β-半乳糖苷酶组成。在这些菌株的粗提物中，色氨酸合成酶α亚基部分可以通过其与β亚基聚集的能力来鉴定，因为一些β亚基活性可以用抗β-半乳糖苷酶的抗血清沉淀。此外，β-半乳糖苷酶也能用抗色氨酸合成酶α亚基的抗血清沉淀。乳糖阻遏物部分能够在体外结合IPTG，但不能结合lac操纵子DNA。β-半乳糖苷酶部分与原始的乳糖阻遏物-β-半乳糖苷酶嵌合体一样不受影响。通过SDS凝胶电泳测定，两种嵌合体的分子量均为175,000。嵌合体部分降解，产生不同分子量的片段。
