Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
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BERVANAKIS, G.Chapter 3: RESULTSStreptomyces <strong>and</strong> Micromonospora, known to conta<strong>in</strong> deoxysugar moieties (Figure28).Gene Organization <strong>of</strong> Deoxysugars ComponentsstrD strE strL strMstrD01fstrD01rPCR product = 0.37 kbStrD = dTDP-glucose synthase strE = dTDP-glucose-4-6-dehydratase strL = dTDPdihydrostreptosesynthase strM = dTDP-4-keto-6-deoxyglucose 3,5-epimeraseFigure 28. Illustration depict<strong>in</strong>g the organization <strong>of</strong> the deoxyhexose genes.Evaluation <strong>of</strong> the strD01f primer am<strong>in</strong>o acid sequence alignments <strong>in</strong>dicates a highlyconserved sequence across different act<strong>in</strong>obacterial species XXXXLGDN [X positionrepresents codon degeneracy’s] (Table 41). This sequence could be used to design aprimer to screen for the strD gene <strong>in</strong> act<strong>in</strong>obacteria. The MSA <strong>of</strong> the primers<strong>in</strong>dicated that they were biased towards Streptomyces species (Table 41).Table 41. Relative am<strong>in</strong>o acid positions <strong>and</strong> codon degenerecies <strong>of</strong> the strD01f <strong>and</strong>strD01r primers <strong>in</strong> the sequence.Primer Am<strong>in</strong>oAcidActualPrimerStreptomycesAm<strong>in</strong>o AcidAm<strong>in</strong>o AcidSequence~Am<strong>in</strong>o AcidSequence*Position Am<strong>in</strong>o AcidSequenceSequence astrD01f 111 - 118 FAMYLGDN FVMYLGDN (X)(X)M(X)LGDN (X)(X)M(X)LGDNstrD01r 182 - 187 YGRTPA YLFTPA Y(X)(X)(X)(X)(X) Y(X)Y(X)(X)D(X) = variable codona Streptomyces consesus sequence for targeted regions~ Streptomycete <strong>and</strong> Non-Streptomycete Act<strong>in</strong>obacteria species* Non - Streptomycete Act<strong>in</strong>obacteria species onlyTable 42. Relative nucleic acid positions <strong>of</strong> the strD01f <strong>and</strong> strD01r primers <strong>and</strong>variable nucleotides with<strong>in</strong> the sequencePrimer NucleicAcidNucleic AcidSequence~Nucleic AcidSequence*PositionstrD01f 326 - 350 CNTNGNNNTGNNNCTNGGNGACAA NNTNNNNNTNNNNCTNGGCGACAAstrD01r 677 - 697 No Consensus a N/C(N) = variable baseN/C - means that there are no corresspond<strong>in</strong>g sequences for a comparison to be madea No consensus implies that sequences were highly variable <strong>and</strong> no conserved regions were detected~ Streptomycete <strong>and</strong> Non-Streptomycete Act<strong>in</strong>obacterial species* Non- Streptomyces species only_____________________________________________________________________89
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