Lightner V A, Larson T J, Tailleur P, Kantor G D, Raetz C R, Bell R M, Modrich P
J Biol Chem. 1980 Oct 10;255(19):9413-20.
Si+ hybrid ColE1 plasmids of the Clarke-Carbon collection (Clarke, C., and Carbon, J. (1976) Cell 9, 91-99) which eliminate the sn-glycerol 3-phosphate growth requirement of a mutant of Escherichia coli with a Km defect in sn-glycerol-3-phosphate acyltransferase (plsB) were identified. Marked overproduction of a plasmid-encoded sn-glycerol-3-phosphate acyltransferase with a wild type Km in a host plsB- background indicates that the hybrid plasmids carry a structural gene for this enzyme. In addition, all of these plasmids suppress the phenotype of a mutation in a second locus involved in phospholipid biosynthesis, dgk (diglyceride kinase), and one of them also bears the dnaB structural gene. Diglyceride kinase activity is also overproduced in these strains. The linkage of plsB, dgk and dnaB loci was confirmed by transduction analysis which demonstrated the clockwise gene order malB, dnaB, dgk, plsB, and uvrA near Minute 91 on the E. coli linkage map. This is in contrast to the previously reported co-transduction of plsB with dctA near Minute 78 (Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227-233). Recloning of restriction endonuclease fragments and in vitro mutagenesis have localized the dgk, and plsB loci to a 2.2-megadalton DNA segment, and have demonstrated that diglyceride kinase and sn-glycerol-3-phosphate acyltransferase activities reside in separate polypeptides. Availability of these clones and mutationally altered derivatives has allowed the identification of a single polypeptide (Mr = 83,000) corresponding to the sn-glycerol-3-phosphate acyltransferase and purification of this membrane-bound enzyme to near homogeneity (Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421-9426). The size of the plsB polypeptide indicates that a major fraction of the DNA segment to which this gene has been localized is involved in coding for the sn-glycerol-3-phosphate acyltransferase.
已鉴定出克拉克 - 卡本文库（Clarke, C., and Carbon, J. (1976) Cell 9, 91 - 99）中的Si + 杂种ColE1质粒，这些质粒可消除在sn - 甘油 - 3 - 磷酸酰基转移酶（plsB）中存在Km缺陷的大肠杆菌突变体对sn - 甘油 - 3 - 磷酸的生长需求。在宿主plsB - 背景中，具有野生型Km的质粒编码的sn - 甘油 - 3 - 磷酸酰基转移酶显著过量表达，这表明杂种质粒携带该酶的结构基因。此外，所有这些质粒都能抑制参与磷脂生物合成的第二个位点dgk（甘油二酯激酶）中的突变表型，其中一个质粒还带有dnaB结构基因。在这些菌株中，甘油二酯激酶活性也过量表达。通过转导分析证实了plsB、dgk和dnaB位点的连锁关系，该分析表明在大肠杆菌连锁图谱上，基因顺序为顺时针方向的malB、dnaB、dgk、plsB和uvrA，位于第91分钟附近。这与之前报道的在第78分钟附近plsB与dctA的共转导情况相反（Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227 - 233）。通过对限制性内切酶片段的再克隆和体外诱变，已将dgk和plsB位点定位到一个2.2兆道尔顿的DNA片段上，并证明甘油二酯激酶和sn - 甘油 - 3 - 磷酸酰基转移酶活性存在于不同的多肽中。这些克隆和经突变改变的衍生物的可用性，使得能够鉴定出一种对应于sn - 甘油 - 3 - 磷酸酰基转移酶的单一多肽（Mr = 83,000），并将这种膜结合酶纯化至接近均一（Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421 - 9426）。plsB多肽的大小表明，该基因所在的DNA片段的大部分参与了sn - 甘油 - 3 - 磷酸酰基转移酶的编码。
