Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University
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Abstract |
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Introduction |
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Hughes, Ota, and Nei (1990)
proposed to circumvent the saturation problem by using the ratio of radical to conservative amino acid replacements. The rationale of this method is very similar to that used in the nonsynonymous-synonymous ratio case. That is, radical replacements are assumed to be more likely than conservative replacements to improve the function of a protein. Therefore, if positive selection plays a major role in the evolution of a protein, we should expect the radical-conservative ratio to exceed the expectation under no selection. There are several methods to estimate the radicalism or conservatism of a particular amino acid replacement. One, for example, may decide that the property of interest is electric charge, and therefore, all replacements that result in charge changes are radical, whereas all replacements that do not affect charge are conservative. Alternatively, several properties may be considered simultaneously through the use of a physico-chemical measure, such as Grantham's (1974)
distance. The radical-conservative replacement ratio has also been used extensively to infer positive selection (e.g., Hughes, Ota, and Nei 1990
; Hughes 1992
; Rand, Weinreich, and Cezairliyan 2000
; Hughes 2000, 2002
).
In this study, we investigate the possibility that factors unrelated to selection influence the radical-conservative replacement ratio values. For example, it is known that transversions result in more dramatic changes than do transitions. That is, transversions are more likely than transitions to be nonsynonymous in protein-coding regions, and nonsynonymous transversions are more likely to result in radical replacement than nonsynonymous transitions (Zhang 2000
). It is, therefore, possible that differences in radical-conservative replacement ratios may be caused by mutations factors, such as the transition-transversion ratio, rather than selectional forces. In this study, we simulated DNA-sequence evolution and resulting radical-conservative replacement ratios by varying transition-transversion ratios, codon usage, genetic code, and amino acid composition. In the simulation we introduced no hint of positive selection.
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Methods |
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Radical-Conservative Ratios
All the 190 possible amino acid replacements were classified using three independent criteria: (1) charge, (2) volume and polarity, and (3) Grantham's (1974)
physico-chemical distance.
Classification by charge was made by dividing the amino acids into three categories: positive (R, H, K), negative (D, E), and uncharged (A, N, C, Q, G, I, L, M, F, P, S, T, W, Y, V).
Classification by volume and polarity was made by dividing the amino acids into six categories: special (C), neutral and small (A, G, P, S, T), polar and relatively small (N, D, Q, E), polar and relatively large (R, H, K), nonpolar and relatively small (I, L, M, V), and nonpolar and relatively large (F, W, Y).
The two classifications above were taken from Zhang (2000)
. We did not use an additional classification in Zhang (2000)
, i.e., polarity, in order to keep the divisions independent of one another. Within each of the two classifications above, amino acid replacements were deemed conservative if they involved exchanges within a category and radical if the exchanges occurred among categories.
As far as Grantham's (1974)
distances are concerned, an amino acid replacement was deemed conservative if the distance value was smaller than 100 and radical otherwise.
Codon Usage
Three patterns of codon usage were used: random, GC biased, and AT biased. In the random pattern, each codon frequency was calculated as the frequency of the amino acid specified by the codon divided by the number of possible codons for the amino acid. In the GC- and AT-biased patterns of codon usage, each codon frequency was calculated as the frequency of the amino acid specified by the codon divided by the number of possible codons ending in GC or AT, respectively.
Amino Acid Composition
Eight amino acid compositions were used. Two compositions were the theoretical equilibrium expectations of two replacement matrices, i.e., Dayhoff's (1978, p. 345)
and JTT (Jones, Taylor, and Thornton 1992
). Five compositions were derived from mean amino acid frequencies in different protein classes: (1) extracellular proteins, (2) anchored proteins, (3) membranal proteins, (4) intracellular proteins, and (5) nuclear proteins. The values were taken from Cedano et al. (1997)
. The eighth composition was of a proline-rich protein as an example of extreme amino acid bias. In this case, the frequency of 19 amino acids was set at 0.045, whereas the frequency of proline was 0.136. All amino acid frequencies are shown in table 1
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Insertion and deletion frequencies were set to zero in order to keep the length of the sequences constant and prevent gaps in the alignment.
Genetic Code
Two genetic codes were used: the standard (so-called universal) code and the vertebrate mitochondrial code.
Statistical Analyses
The effects of various variables and the interactions among them on the three radical-conservative replacement ratios were tested by a multiway analysis of variance (ANOVA). All the effects were considered as fixed.
Reality check
In order to establish that compositional and mutational factors may indeed produce false positive inferences of Darwinian selection, we simulated the evolution of several human protein-coding genes in which positive selection has never been reported, e.g., ß hemoglobin, interleukin 2, ribosomal protein S21 (accession numbers NM_000518.3, NM_001024.2, and NM_000586.1, respectively) under the substitution matrix of pseudogenes (presumably a completely neutral matrix of substitution reflecting the pattern of mutation without selection). The neutral substitution matrix was taken from Graur and Li (1999
, p. 126)
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Results and Discussion |
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We conclude that many factors that have nothing to do with selection (positive or otherwise) either singly or in combination affect measures that were supposed to be indicative of positive selection. Therefore, selectional inferences based on radical-conservative replacement ratios should be treated with utmost caution. In fact, we recommend that these measures not be used at all.
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Footnotes |
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Address for correspondence and reprints: Tal Dagan, Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. tali{at}kimura.tau.ac.il
.
Keywords: positive Darwinian selection
conservative replacement
radical replacement
transition bias
codon usage
genetic codes
amino acid composition
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References |
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