Lilley P E, Stamford N P, Vasudevan S G, Dixon N E
Centre for Molecular Structure and Function, Research School of Chemistry, Australian National University, Canberra, ACT.
Gene. 1993 Jul 15;129(1):9-16. doi: 10.1016/0378-1119(93)90690-5.
A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation. Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes. Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320. Mutant complementation by plasmids showed that ubiA is located between malM and plsB. A previously sequenced long open reading frame that encodes the C-terminal portion of the E. coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product). Comparison of homologous regions from E. coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB. Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx. 40-kDa protein with ARase activity.
通过选择对dnaB突变进行互补作用，分离出了一个携带42.5 kb大肠杆菌染色体DNA的黏粒（pND320），其中包括连锁图谱上第92分钟附近xylE和ssb之间的基因。利用已知的核苷酸（nt）序列将该区域的限制酶图谱与染色体物理图谱（坐标4319.5至4362 kb）进行比对，并精确确定和定义了19个基因的方向。通过对从pND320亚克隆的片段进行nt序列分析，证实了已测序基因在确定长度的未测序间隙上的预测物理连锁关系。质粒的突变互补作用表明ubiA位于malM和plsB之间。一个先前已测序的长开放阅读框编码大肠杆菌ubiA产物（对羟基苯甲酸聚异戊二烯基转移酶，HPTase）的C末端部分，它与酵母HPTase（COQ2基因产物）的相应片段具有高度的序列同一性。利用大肠杆菌和鼠伤寒沙门氏菌同源区域的比较，精确地将编码丙氨酸消旋酶（ARase）的alr基因定位在dnaB和tyrB之间。将alr亚克隆到串联噬菌体λ启动子下游，产生了一个质粒，该质粒可指导一种具有ARase活性的可溶性约40 kDa蛋白的高水平过量表达。
