Mutants were confirmed by PCR and Southern hybridization. Tests of Dnd phenotype were described in [5, 8] or [10, 15]. Table 1 primers used in PCR and RT-PCR Primer Name Sequence (with the restriction enzyme sites underlined) Enzyme site A2 ATCACCCCTTCCACCGAGAT A1 ACTGGATGACCGCGGAGTTC B1 GAGTACGTTTTTCCGGCCATCC B2 TCCTTCAGCGCCTGCTCGAT B3 CCAACACCGACTGGGAGGGG C1 CAGAGATCGTCGAGGAGCTG C2 GATCTTCAACCGCTCGGTGC C3 CAGTATCGAACCATGACCCGG D1 TGCGGCAAGACGACCCTGCT D2 GTCGGCGAGCTGTTCCACCT D3 CAGTGATCGACACCCCACTC E1 ATGCCGTCTGAGATCACCAT E2 ATAAGCAGCGTCTTGCCCAC 16S rRNA SP
AGTAACACGTGGGCAACTGC 16S rRNA this website AP CTCAGACCAGTGTGGCCGGT xtg1 CCGATCTTGTGCCCGCTGATG xtg2 GCGCCTTAAGTCGTCCCTTGTTC AflII xtg3 GAAGGTGTCTTAGATCTCCGG BglII xtg4 CTGGCACGACAGGTTTCC xtg5 AAGCACCGGTTCAAGACG AgeI xtg6 GCCCAGGTCCGCAAGAA xtg7 CTCGTGGTTGAGCGGGACTACGG xtg8 CTGGCACCGGTCAAGCCTAGGTG AgeI, AvrII xtg9 GGGACAGCCTAGGGGTGATC AvrII xtg10 ACTGACCGCAGACCGCAAG wlr5 CATATGGTGGGATCTTCTGCAGCT NdeI wlr6 GGATCCTCAATGATGATGATGATGATGTGACTCTCCTCGCAGGTA BamHI wlr7 CATATGAGCACCCCCAAGGCG NdeI wlr11 GGATCCTTAGTGGTGGTGGTGGTGGTGTGCAGGTGCATCGGTGGTGA BamHI
dnd-1 AGAGATCACCACATATGCACCTGAGCACC NdeI dnd-2 CAGCCGGATCCTGATCTCAG BamHI dndE-L CACATATGCCGTCTGAGATCACC NdeI dndE-R TAAGGCCTATTCGGCGGTGA Intensity of DNA bands was quantified from the fluorescence intensity using GeneTool software (Syngene). Refinement of the limits of the dnd gene cluster pHZ1900: a 10-kb BamHI fragment from
pHZ825 was cloned selleck chemical into pSET152. Progesterone pJTU1203 or pJTU1204 (with opposite direction): a 7.9-kb MluI-EcoRI fragment from pHZ1904 was blunt-ended and cloned into the EcoRV site of pSET152. pJTU1208: the 1.0-kb BglII fragment from pHZ1900 was inserted into the BamHI site of pBluescript II SK (+). Then a 0.3-kb SalI fragment of this plasmid was replaced with a 1.3-kb SalI fragment from pHZ1904 to MK-0457 manufacturer generate pHZ2850, in which dndA accommodated in a 2.0-kb BamHI/BglII-SacI region. A 1.4-kb fragment from pHZ2850 generated by complete digestion with EcoRI and partial digestion with BglII was inserted into the EcoRI and BamHI sites of pSET152 to give pHZ2851. Finally, a 2.1-kb XbaI-SfiI fragment of pJTU1204 was replaced with a corresponding 0.8-kb fragment from pHZ2851, generating pJTU1208. Thus, in pJTU1208, the dnd gene cluster was shortened to the BglII site near the end of dndA, covering a 6,665-bp region. pHZ2862 (also the vector for dndA deletion): a 2.0-kb PvuII fragment from pHZ1900 was cloned into the SmaI site of pBluescript II SK(+) to give pHZ2853, then a 6.5-kb SmaI-EcoRI fragment from pHZ1900 was used to replace the 0.7-kb corresponding fragment in pHZ2853 to give pHZ2861, in which dndB-E lay in a 7.8-kb SmaI/PvuII-EcoRI region. A 7.8-kb BamHI fragment from pHZ2861 was cloned into pSET152 to give pHZ2862.