Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium.
The glnA gene, encoding glutamine synthetase in Salmonella typhimurium, has been cloned into the plasmid pBR322. One hybrid plasmid, pJB1, containing an 8.5 kb insert generated by a HindIII digest, was analyzed using eleven different restriction enzymes. Evidence that the region controlling glutamine synthetase expression remained on the insert was obtained by showing that the regulation is normal in cells carrying plasmids with the insert in the original and reversed orientation. Several new plasmids derived from pJB1 following SalI and EcoRI digestions were examined for their ability to complement a glnA202 mutation in order to locate the DNA segment needed for glutamine synthetase expression. The results show that cells containing plasmid pJB8, which has a 21 kb deletion, produce and regulate glutamine synthetase normally, whereas cells with a plasmid (pJB11) similar to pJB8, but lacking a 0.25 kb EcoRI fragment, do not exhibit glutamine synthetase activity. The analysis of proteins produced in minicells containing pJB8 and pJB11 show that they both produce a protein that migrates with the glutamine synthetase subunit. Because pJB11 makes an inactive protein of similar size to the glutamine synthetase subunit, the 0.25 kb deletion may encode only the C-terminus of this protein. Consistent with this finding is the presence of a strong RNA polymerase-binding site on pJB8 to the right of the 0.25 kb EcoRI that could correspond to a promoter near the N-terminus of the glnA gene.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D012318:RNA Polymerase I / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*
D012318:RNA Polymerase I; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D005974:Glutamate-Ammonia Ligase