Cloning of phosphoenolpyruvate carboxylase gene from a cyanobacterium, Anacystis nidulans, in Escherichia coli.
The phosphoenolpyruvate carboxylase gene (ppc) from Anacystis nidulans, a cyanobacterium (blue-green alga), was cloned in Escherichia coli. Chromosomal DNA of A. nidulans was partially digested with Sau3AI, and the obtained DNA fragments were ligated in the BamHI site of pBR322. The hybrid plasmids were first transformed into E. coli K802 (hsdR-, hsdM+) to obtain the gene bank of A. nidulans. The bank consisted of about 12,000 clones. These hybrid plasmids were then transformed into E. coli PCR1 (ppc2-, recA1-, hsdR+, hsdM+), and the transformants were selected by complementation of the ppc mutation (phenotype of glutamate requirement). In the cell-free extracts of E. coli strains having the cloned ppc gene, PEPCase activities were detected, but their properties were different from those of the E. coli enzyme. Analysis by subcloning showed that the ppc gene was included in a DNA fragment 3,500 base pairs long and the maxicell method revealed that the molecular weight of the gene product was about 108,000. It is suggested that the ppc gene is expressed in E. coli mainly by read-through transcription, being initiated by the promoter of tetracycline-resistance gene of pBR322, but the significant expression in reversed orientation of the cloned ppc gene indicates that the gene includes a promoter capable of functioning in E. coli cells.
Kodaki
T
T
Katagiri
F
F
Asano
M
M
Izui
K
K
Katsuki
H
H
D002262:Carboxy-Lyases / Q000235:genetics*; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics*; D004247:DNA; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D009693:Nucleic Acid Hybridization; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics*; D010957:Plasmids; D014169:Transformation, Bacterial
D004247:DNA; D004262:DNA Restriction Enzymes; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase