The structure of the Escherichia coli hemB gene.
The Escherichia coli hemB gene, which encodes 5-aminolevulinic acid dehydratase, and was cloned into pTZ18U, a multicopy plasmid, was sequenced. The hemB insert was double-digested with restriction enzymes and recloned back into pTZ18U and pTZ19U to allow for sequencing in two directions. In a second procedure, used to fill in gaps and to confirm the sequence derived from the first procedure, the whole insert was cloned into M13 phages. A nested set of deletions was constructed and recloned into M13. Both the double-digested fragments cloned into plasmids pTZ18U and pTZ19U and the overlapping fragments contained in M13 phages were sequenced using the dideoxy procedure with [35S]dATP. Computer software was used to identify coding regions and the correct reading frame. Two promoter regions, two Shine-Dalgarno sequences and two possible start sites were identified. Extensive homologies with yeast (36%), human liver (40%) and rat liver (40%) amino-acid (aa) sequences were observed, especially in the 16-aa Zn-binding region (75%) and the 4 aa surrounding the essential lysine at the active site (100% for rat and human proteins). Computer analysis of promoter strength and two independent analyses of codon usage indicated that the hemB gene is moderately expressed.
Li
J M
JM
Department of Biochemistry, City College, City University of New York.
Russell
C S
CS
Cosloy
S D
SD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D000623:Porphobilinogen Synthase / Q000235:genetics*; D011401:Promoter Regions, Genetic
D003062:Codon; D000623:Porphobilinogen Synthase