Enumerating SIV-specific CD8-TL responses by IFN-γ ELISPOT.
O'Connor in Lasso dynamic markup language (Blueworld, Bellevue, Wash.). These nucleotide alignments were conceptually translated into amino acid alignments that distinguish mixed-base substitutions from complete substitutions by use of a script written by D.
#REMOVE DASHED BOX IN SEQUENCHER OUTPUT TRIAL#
Nucleotide sequences were aligned with the wild-type SIVmac239 sequence in MacVector 7.2, trial version (Accelrys, San Diego, Calif.). Sequences were edited with Sequencher 4.1 (Genecodes, Ann Arbor, Mich.), and mixed-base polymorphisms were identified automatically by Sequencher. The sequences were run on either an ABI 377 or ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, Calif.). Mixed populations of sequences with different lengths, particularly in env, could not be resolved by direct sequencing and are annotated with “X's” in Fig. An additional set of eight PCR primer pairs was used to amplify problematic regions of the genome. Viruses were sequenced at the time of host death as described previously ( 38), with slight modifications.
Undetermined bases, which likely resulted from insertion-deletion replacements, were excluded from the analyses. The property of statistical independence enabled us to use statistical methods that are not dependent on assumptions regarding the substitution model and thus have more robustness than model-based methods that are typically applied for analyses of nucleotide substitution patterns. Because the virus from each monkey was compared independently with the inoculum, each comparison was phylogenetically ( 17) and thus statistically independent. In the case of ambiguous nucleotides, we assumed equal occurrences of the possible nucleotides in the viral population in any given monkey.
In preliminary analyses, more complex models of nucleotide substitution ( 28, 50) yielded identical results, as expected when (as in the present data) the number of substitutions per site is low ( 36). Since the Vif 100-107VI8 CD8-TL epitope was identified partly based on variation data from this study, it was excluded from statistical analyses. The number of synonymous substitutions per synonymous site ( d S) and the number of nonsynonymous substitutions per nonsynonymous site ( d N) were estimated by the method of Nei and Gojobori ( 35) for the sequence obtained from each virus and for the inoculum sequence.
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