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2642687254
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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0029125339
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Hutchinson, C.R.5
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2642700574
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note
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The 10,000g supernatant from a cell lysate of M. tuberculosis H37Rv that had been treated with 1 to 5 μg/ml INH was brought to 80% saturation with solid ammonium sulfate. The supernatant from centrifugation at 10,000g was acidified with acetic acid to pH 3.9, centrifugea, and the pellet was redissolved in distilled water. Further purification was accomplished by preparative native gel electrophoresis followed by electroelution.
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2642698465
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35S]methionine, and after 4 hours, the bacteria were harvested by centrifugation and resuspended in an equal volume of water. This was added to10 g of 0.1-mm glass beads and homogenized at 4°C for three 1-min pulses by bead-beating and centrifuged at 10,000g for 15 min. The 70 and 80% ammonium sulfate pellets were resuspended in 10 ml of 50 mM sodium phosphate (pH 6.0) containing 0.75 M ammonium sulfate and loaded onto an HP1610 Phenyl-HIC column (Pharmacia). This column was eluted from a concentration of 0.75 M ammonium sulfate until it reached 0 M. Fractions containing the 80-kD protein, determined by protein immunoblot of unlabeled-samples, or radioactivity, were combined and dialyzed against 10 mM MES (pH 6.0) before loading onto a Resource Q column equilibrated in the same buffer. After a 2-column volume (CV) wash, the column was eluted over 10 CV to 36% 1 M NaCl in 10 mM MES (pH 6.0) (B), and then to 42% B over another 20 CV, and finally to 100% B over another 10 CV. Appropriate fractions were pooled and concentrated.
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0029927505
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A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68, 850 (1996).
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Shevchenko, A.1
Wilm, M.2
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Mann, M.4
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2642654879
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note
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6 colonies/μg of DNA. kasA was polymerase chain reaction-amplified using Pwo polymerase and was cloned into pMH29H, in which transcription is driven by a synthetic mycobacterial promoter sequence. Transformation of this construct into M. smegmatis and M. tuberculosis produced very small slowly growing colonies. These colonies overproduced KasA protein as demonstrated by SDS-PAGE and protein immunoblotting using affinity-purified antisers to a peptide segment of the KasA primary sequence. It was not possible to accurately assess the INH susceptibility of these colonies because of their impaired growth, but they did not appear to be significantly more resistant to INH than were controls.
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2642657952
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315 mutation, four copies of IS6110, and belonged to group 1. Strain HN93 was recovered from a lymph-node biopsy of a Houston patient and showed INH resistance at 1 to 2 μg/ml. This strain had a single copy of IS6110 and also belonged to group 1. None of these strains had alterations in the inhA locus or upstream of ahpC.
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We thank H. Caldwell for proofreading the manuscript and the NIH-AIDS Research and Reference Reagent Program of NIAID for providing radioactive INH. Supported by USPHS grant AI37004 to J.M.M.
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