Abstract
The gene coding for the biosynthetic thiolase from Zoogloea ramigera has been isolated by using antibody screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter. We have located and determined the nucleotide sequence of the gene. The structural gene is 1173 nucleotides long and codes for a polypeptide of 391 amino acids; 282 nucleotides 5' and 58 nucleotides 3' to the coding sequence are also reported. By comparing the amino acid sequence data predicted from the gene with data determined experimentally, we have derived the complete primary structure of thiolase. A catalytically essential cysteine is located at residue 89. The DNA sequence presented has a very high G/C content, 66.2%, typical of the Z. ramigera genome. In the coding region, this increases to 68.2% and is strongly reflected in the codon usage which demonstrates a strong preference for G or C in the third position. Examination of the 5'-flanking sequence establishes that the NH2-terminal methionine is specified by an ATG codon, 7 nucleotides downstream from a Shine-Dalgarno sequence.
Highlights
Screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter
By comparing the amino acid sequence data predicted from the gene with data determinedexperimentally, we have derived the complete primary structure of thiolase
Multiple formsof thiolases have been isolated from a number of sources where they are involved in the @oxidation of fatty acids,ketogenesis, and cholesterogenesis
Summary
Screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter. By comparing the amino acid sequence data predicted from the gene with data determinedexperimentally, we have derived the complete primary structure of thiolase. Using this approachw, e have isolated a recomthat the NHz-terminal methionine is specified by an binant clone containing thecomplete thiolase gene sequence. DNA insert in this clone and sequenced the entire thiolase gene-coding region. By using protein sequence data for the NHz-terminal 25 amino acids and a10-aminoacid tryptic. Zoogloea ramigera 1-16” is a floc-formingbacteria isolated from activated sludge [1].This organism plays an important role in waste water treatment bothby its ability tolower the biological oxygen demand and by inducing the formation of peptidefromtheactivesite of theprotein[39], we have determinedthecorrectreadingframe for translationand the complete primary amino acid sequence of the protein
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