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1842342533
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note
-
For the first round of PCR, we used cDNA from P. homomalla total RNA and the primers 5′-GGTTCCAARTGGYTNATGGCNAA and 5′-CTATGTRTGIATRCTRTTIGGIAT (7). For the second round of PCR, we used the equivalent of 0.1 μl of the first-round PCR products as template with the same upstream primer and the nested downstream primer 5′-CCAGATCAGIAAICCRTCRTCICKRTA. The 405-bp product included sequences from the previously identified 8R-lipoxygenase (7) and the lipoxygenase described here.
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15
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1842314699
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The 5′ end of the sequence was cloned with the Marathon cDNA Amplification kit (Clontech) (7) and the 5′ RACE procedure (Gibco-BRL)
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The 5′ end of the sequence was cloned with the Marathon cDNA Amplification kit (Clontech) (7) and the 5′ RACE procedure (Gibco-BRL).
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16
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0027246677
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note
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4 tag) was expressed in the pET3a vector in Escherichia coli (BL21, Novagen) incubated at 28°C in TB medium for 24 hours. This resulted in appearance in the bacterial cytosolic fraction of a hemoprotein detectable by UV-visible spectroscopy (≈1 absorbance unit at 406 nm) that was not present in cells transfected with vector alone. The enzyme was purified by ammonium sulfate precipitation (30 to 55% fraction) and by anion exchange chromatography on Q-Sepharose, and on a nickel affinity column (Qiagen) for the His-tagged protein.
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21
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1842302416
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2-terminus from the 5′ end to nucteotide 1116 of the ORF
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2-terminus from the 5′ end to nucteotide 1116 of the ORF.
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22
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We thank W. Boeglin for help with the HPLC analyses and J. Swanson and his colleagues at the Keys Marine Laboratory for collection of the P. homomalla specimens. Supported by a grant from NIH (GM49502) and a Fogerty International Research Collaboration Award (TW00404)
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We thank W. Boeglin for help with the HPLC analyses and J. Swanson and his colleagues at the Keys Marine Laboratory for collection of the P. homomalla specimens. Supported by a grant from NIH (GM49502) and a Fogerty International Research Collaboration Award (TW00404).
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