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Title: Nucleotide analysis of two actinomycete aminoglycoside resistance determinants
Author: Holmes, David John
ISNI:       0000 0001 3580 7398
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1989
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Resistance to aminoglycosides in the organisms that produce them is often ascribed to the well characterised and clinically important antibiotic modifying enzymes. However, at least three aminoglycoside producing actinomycetes, namely Micromonospora purpurea, Streptomyces tenjimariensis, and Streptomyces tenebrarius possess ribosomes that are refractory to some members of this class of drugs. In these cases, resistance is due to methylation of rRNA of the small ribosomal subunit. This study supports the possibility that this mechanism might be more widespread than hitherto suspected. Two of the methylase genes have been analysed at the nucleotide level and their transcripts mapped. The gentamicin resistance methylase gene (kgmA) from M. purpurea codes for a 36 kDa protein consisting of 249 amino acids. Like most actinomycete genes, kgmA is not expressed in E. coli from its own promoter, although the determinant was expressed in this Gram-negative host as a result of DNA rearrangement. Sequence analysis of the mutated plasmid suggested that the methylase was expressed as a translational fusion with the lacZ' gene of pUC18, a view that was later confirmed. Transcript mapping revealed that kgmA is probably read from a single promoter but that it might be part of a polycistron. The second gene examined confers resistance to kanamycin and apramycin, and originated in S. tenjimariensis. This determinant (kamA) was shown to encode a predicted protein of 155 amino acids with a molecular weight of 19 kDa. Unlike kgmA, this gene could not be expressed as either a transcriptional or translational fusion in E. coli. Transcription of kamA is directed by tandem promoters and is a monocistron since the the transcript terminates only 160 bp downstream of the stop codon.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Gene cloning in Streptomyces