Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 3: RESULTSDespite this the strD01f and strD01r primers bias towards Streptomyces speciesproduced partial bands of expected size in both the Micromonospora species tested.This indicated that a similar sequence was present in the Micromonospora speciesgenome (Figure 29). Additional extra bands of unexpected size were detected whichmay have resulted from partial homologies between primers and template DNA(Figure 29:lane 4).(kb) 1 2 3 4 52.01.51.00.70.50.40.30.20.1Figure 29. Agarose gel electrophoresis of 0.37 kb strD segment amplified DNAfrom aminoglycoside positive actinobacterial type cultures. Lanes 1: Molecularweight marker in kilobases (kb). Lane 2: S. coelicolor M145 DNA amplified16SrDNA region. Lanes 3: Streptomyces griseus (DSM40236), Lane 4:Micromonospora purpurea (DSM43036), Lane 5: Micromonospora olivasterospora(ATCC21819).Direct sequencing of the amplified 0.37 kb product from the pure strain Streptomycesgriseus DSM40236, known to contain the strD gene (Distler et al., 1987) confirmedthat the correct gene had been amplified. A Gapped-FASTA database search revealedthat both the nucleotide and translated amino acid sequences derived from this typestrain showed sequence similarities (58 – 83%) and (54 - 80%), respectively, toknown dTDP-glucose synthase genes and their product in actinobacteria (Table 43).The PCR product obtained with the strD01f and strD01r primers indicated thatputative dTDP-glucose synthase genes could be detected using this primer pair for theCerylid isolates. This was indicated by the PCR products of the predicted length_____________________________________________________________________90