Evolution of protein inhibitors of serine proteinases: Positive Darwinian selection or compositional effects?

Summary In at least two instances involving serine proteinase inhibitors it has been shown that functionally important sites evolve faster and exhibit more interspecific variability than functionally neutral sites. Because these phenomena are difficult to reconcile with the neutral theory of molecular evolution, it has been suggested that the accelerated rate of amino acid substitution at the reactive sites is brought about by positive Darwinian selection. We show that differences in the amino acid composition in the different regions of proteinase inhibitors can account for the differences in the rates of amino acid substitution. By using an index of protein mutability [D. Graur (1985) J Mol Evol 2253–62], we show that the amino acid composition of the reactive center in the ovomucoids andSpi-2 gene products is such that, regardless of function, they are expected to evolve more rapidly than any other polypeptide for which the rate of substitution is known. In addition, the reactive region in theSpi-2 proteins is shown to be free of compositional constraint. Positive Darwinian selection need not be invoked at the present time in these cases.     

Mammalian gene evolution: Nucleotide sequence divergence between mouse and rat

As a paradigm of mammalian gene evolution, the nature and extent of DNA sequence divergence between homologous protein-coding genes from mouse and rat have been investigated. The data set examined includes 363 genes totalling 411 kilobases, making this by far the largest comparison conducted between a single pair of species. Mouse and rat genes are on average 93.4% identical in nucleotide sequence and 93.9% identical in amino acid sequence. Individual genes vary substantially in the extent of nonsynonymous nucleotide substitution, as expected from protein evolution studies; here the variation is characterized. The extent of synonymous (or silent) substitution also varies considerably among genes, though the coefficient of variation is about four times smaller than for nonsynonymous substitutions. A small number of genes mapped to the X-chromosome have a slower rate of molecular evolution than average, as predicted if molecular evolution is male-driven. Base composition at silent sites varies from 33% to 95% G + C in different genes; mouse and rat homologues differ on average by only 1.7% in silent-site G + C, but it is shown that this is not necessarily due to any selective constraint on their base composition. Synonymous substitution rates and silent site base composition appear to be related (genes at intermediate G + C have on average higher rates), but the relationship is not as strong as in our earlier analyses. Rates of synonymous and nonsynonymous substitution are correlated, apparently because of an excess of substitutions involving adjacent pairs of nucleotides. Several factors suggest that synonymous codon usage in rodent genes is not subject to selection.     

Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application

Summary A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of øX174 and G4 genomes, histone genes and-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of øX174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between øX174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the nonoverlapping segment. It is also suggested that, in the coding regions surrounding the splicing points of intervening sequences of-globin genes, there exist rigid secondary structures. It is in only these regions that the-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.     

The rates of evolution in some ribosomal components

Summary The rate of nucleotide substitution (k(nuc)) of 5s RNA was estimated to be (1.8 ± 0.5) × 10^–10 per site per year by comparing the nucleotide sequences of human andXenopus 5s RNA and using the geological time elapsed since the separation of mammals and amphibians. Similarly, k(nuc) of 5.8s rRNA was calculated to be 0.93 – 1.4 × 10^–10 per site per year from the sequences of rat hepatoma cells andSaccharomyces cerevisiae. For the comparison of these data with the amino acid substitution rate of known proteins, the k(nuc) values of 5s rRNA and 5.8s rRNA were converted to the rate of amino acid substitution (k(aa)). The k(aa) values in pauling units were 0.4 and 0.2 – 0.3, respectively.The average k(aa) of ribosomal proteins was also estimated to be 0.2 – 0.3 pauling from the N-terminal amino acid sequences of seventeen 30s ribosomal proteins ofBacillus stearothermophilus andEscherichia coli. Thus, the evolutionary rates of these ribosomal components studied here are similar to each other; they are considerably slower than that of the known cellular proteins. Most, if not all, of the replacements in ribosomal proteins occurred between amino acids of a chemically similar nature.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Direct correlation between proteins' folding rates and their amino acid compositions: an ab initio folding rate prediction

Discovering the mechanism of protein folding, in molecular biology, is a great challenge. A key step to this end is to find factors that correlate with protein folding rates. Over the past few years, many empirical parameters, such as contact order, long-range order, total contact distance, secondary structure contents, have been developed to reflect the correlation between folding rates and protein tertiary or secondary structures. However, the correlation between proteins' folding rates and their amino acid compositions has not been explored. In the present work, we examined systematically the correlation between proteins' folding rates and their amino acid compositions for two-state and multistate folders and found that different amino acids contributed differently to the folding progress. The relation between the amino acids' molecular weight and degeneracy and the folding rates was examined, and the role of hydrophobicity in the protein folding process was also inspected. As a consequence, a new indicator called composition index was derived, which takes no structure factors into account and is merely determined by the amino acid composition of a protein. Such an indicator is found to be highly correlated with the protein's folding rate (r > 0.7). From the results of this work, three points of concluding remarks are evident. (1) Two-state folders and multistate folders have different rate-determining amino acids. (2) The main determining information of a protein's folding rate is largely reflected in its amino acid composition. (3) Composition index may be the best predictor for an ab initio protein folding rate prediction directly from protein sequence from the standpoint of practical application.     

Molecular evolution of nuclear genes in Cupressacea, a group of conifer trees

We surveyed the molecular evolutionary characteristics of 11 nuclear genes from 10 conifer trees belonging to the Taxodioideae, the Cupressoideae, and the Sequoioideae. Comparisons of substitution rates among the lineages indicated that the synonymous substitution rates of the Cupressoideae lineage were higher than those of the Taxodioideae. This result parallels the pattern previously found in plastid genes. Likelihood-ratio tests showed that the nonsynonymous-synonymous rate ratio did not change significantly among lineages. In addition, after adjustments for lineage effects, the dispersion indices of synonymous and nonsynonymous substitutions were considerably reduced, and the latter was close to 1. These results indicated that the acceleration of evolutionary rates in the Cupressoideae lineage occurred in both the nuclear and plastid genomes, and that generally, this lineage effect affected synonymous and nonsynonymous substitutions similarly. We also investigated the relationship of synonymous substitution rates with the nonsynonymous substitution rate, base composition, and codon bias in each lineage. Synonymous substitution rates were positively correlated with nonsynonymous substitution rates and GC content at third codon positions, but synonymous substitution rates were not correlated with codon bias. Finally, we tested the possibility of positive selection at the protein level, using maximum likelihood models, assuming heterogeneous nonsynonymous-synonymous rate ratios among codon (amino acid) sites. Although we did not detect strong evidence of positively selected codon sites, the analysis suggested that significant variation in nonsynonymous-synonymous rate ratio exists among the sites. The most likely sites for action of positive selection were found in the ferredoxin gene, which is an important component of the apparatus for photosynthesis.     

Are Radical and Conservative Substitution Rates Useful Statistics in Molecular Evolution?

A DNA mutation in a protein coding gene which causes an amino acid change can be classified as conservative or radical depending on the magnitude of the physicochemical difference between the two amino acids: radical mutations involve larger changes than conservative mutations. Here, I examine two key issues in determining whether radical and conservative substitution rates are useful statistics in molecular evolution. The first issue is whether such rates can be estimated reliably, and for this purpose I demonstrate considerable improvements achieved by simple modifications to an existing method. The second issue is whether conservative and radical substitution rates can tell us something about selection on protein function. I address this problem by estimating positive and negative selection on conservative and radical mutations using polymorphism and divergence data from Drosophila. These analyses show that negative selection, but not positive selection, differs significantly between conservative and radical mutations. The power of conservative and radical substitution rates in testing the nearly neutral theory of molecular evolution is illustrated by the analysis of two mammalian datasets.     

Human triosephosphate isomerase: Substitution of Arg for Gly at position 122 in a thermolabile electromorph variant,TPI-Manchester

Summary Denaturing gradient gel electrophoreses of polymerase chain reaction amplified DNA products and subsequent direct sequencing identified a G-to-A transition causing a replacement of Gly 122 with Arg in an electrophoretic mobility variant of human triosephosphate isomerase, TPI-Manchester. This was the only TPI electromorph variant detected in screening of > 3,400 humans in an Ann Arbor, Mich. population. This substitution is at the amino terminus or solvent interaction end of the fifth sheet of the / barrel structure. The TPI-Manchester variant is á thermolabile enzyme, but the stability of the variant enzyme is not sensitive to other denaturants. This amino acid substitution does not involve residues of the active site and does not detectably alter the kinetic properties of the enzyme. The data provide additional insight into the amino acid residues that are important for the maintenance of the structural characteristics of this very evolutionary constrained protein.     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories (<Emphasis Type="Italic">Ipomoea</Emphasis>)

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoides, which are important for the pigmentation of flowers and act as attractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morning glories (Ipomoea), five functional CHS genes (A–E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B, and C experienced accelerated rates of amino acid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories underwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/synonymous rate ratio ( = d_N/d_S) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test suggested significant variation in selection pressure among amino acid sites, with a small proportion of them detected to be under positive selection along the branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergence of subfamily ABC and subfamily DE and is at least partially responsible for a rate increase following gene duplication.     

Two types of amino acid substitutions in protein evolution

Summary The frequency of amino acid substitutions, relative to the frequency expected by chance, decreases linearly with the increase in physico-chemical differences between amino acid pairs involved in a substitution. This correlation does not apply to abnormal human hemoglobins. Since abnormal hemoglobins mostly reflect the process of mutation rather than selection, the correlation manifest during protein evolution between substitution frequency and physico-chemical difference in amino acids can be attributed to natural selection. Outside of abnormal proteins, the correlation also does not apply to certain regions of proteins characterized by rapid rates of substitution. In these cases again, except for the largest physico-chemical differences between amino acid pairs, the substitution frequencies seem to be independent of the physico-chemical parameters. The limination of the substituents involving the largest physicochemical differences can once more be attributed to natural selection. For smaller physico-chemical differences, natural selection, if it is operating in the polypeptide regions, must be based on parameters other than those examined.     

Mutational bias affects protein evolution in flowering plants

Amino acid sequences from several thousand homologous gene pairs were compared for two plant genomes, Oryza sativa and Arabidopsis thaliana. The Arabidopsis genes all have similar G+C (guanine plus cytosine) contents, whereas their homologs in rice span a wide range of G+C levels. The results show that those rice genes that display increased divergence in their nucleotide composition (specifically, increased G+C content) showed a corresponding, predictable change in the amino acid compositions of the encoded proteins relative to their Arabidopsis homologs. This trend was not seen in a "control" set of rice genes that had nucleotide contents closer to their Arabidopsis homologs. In addition to showing an overall difference in the amino acid composition of the homologous proteins, we were also able to investigate the biased patterns of amino acid substitution since the divergence of these two species. We found that the amino acid exchange matrix was highly asymmetric when comparing the High G+C rice genes with their Arabidopsis homologs. Finally, we investigated the possible causes of this biased pattern of sequence evolution. Our results indicate that the biased pattern of protein evolution is the consequence, rather than the cause, of the corresponding changes in nucleotide content. In fact, there is an even more marked asymmetry in the patterns of substitution at synonymous nucleotide sites. Surprisingly, there is a very strong negative correlation between the level of nucleotide bias and the length of the coding sequences within the rice genome. This difference in gene length may provide important clues about the underlying mechanisms.     

Widespread Adaptive Evolution in the Human Immunodeficiency Virus Type 1 Genome

We investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured by using the nonsynonymous/synonymous substitution rate ratio ( = d_N/d_S). To identify amino acid sites under positive selection with > 1, we applied maximum likelihood models that allow variable ratios among sites to analyze genomic sequences of 26 HIV-1 lineages including subtypes A, B, and C. Likelihood ratio tests detected sites under positive selection in each of the major genes in the genome: env, gag, pol, vif, and vpr. Positive selection was also detected in nef, tat, and vpu, although those genes are very small. The majority of positive selection sites is located in gp160. Positive selection was not detected if was estimated as an average across all sites, indicating the lack of power of the averaging approach. Candidate positive selection sites were mapped onto the available protein tertiary structures and immunogenic epitopes. We measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. Our results demonstrate genomewide diversifying selection acting on the HIV-1.     

Individual amino acid balances in young lean and obese Zucker rats fed a cafeteria diet

The amino acid composition of the diet ingested by reference and cafeteria diet-fed lean and obese Zucker rats has been analyzed from day 30 to 60 after birth. Their body protein amino acid composition was measured, as well as the urinary and faecal losses incurred during the period studied. The protein actually selected by the rats fed the cafeteria diet had essentially the same amino acid composition as the reference diet. The mean protein amino acid composition of the rat showed only small changes with breed, age or diet.Cafeteria-fed rats had a higher dietary protein digestion/absorption efficiency than reference diet-fed rats. Obese rats wasted a high proportion of dietary amino acids when given the reference diet, but not on the cafeteria diet. In all cases, the amino acids lost as such in the urine were a minimal portion of available amino acids.In addition to breed, the rates of protein accretion are deeply influenced by diet, but even more by the age — or size — of the animals: cafeteria-fed rats grew faster, to higher body protein settings, but later protein accrual decreased considerably; this is probably due to a limitation in the blueprint for growth which restricts net protein deposition when a certain body size is attained. Obese rats, however, kept accuring protein with high rates throughout.Diet composition — and not protein availability or quality-induced deep changes in amino acid metabolism. Since the differences in the absolute levels of dietary protein or carbohydrate energy ingested by rats fed the reference or cafeteria diets were small, it can be assumed that high (lipid) energy elicits the changes observed in amino acid metabolism by the cafeteria diet. The effects induced in the fate of the nitrogen ingested were more related to the fractional protein energy proportion than to its absolute values. Cafeteria-fed rats tended to absorb more amino acids and preserve them more efficiently; these effects were shown even under conditions of genetic obesity.There were deep differences in handling of dietary amino acids by dietary or genetically obese rats. The former manage to extract and accrue larger proportions of their dietary amino acids than the latter. The effects of both models of amino acid management were largely additive, suggesting that the mechanisms underlying the development of obesity did not run in parallel to those affecting the control of amino acid utilization. Obesity may be developed in both cases despite a completely different strategy of amino acid assimilation, accrual and utilization. (Mol Cell Biochem121: 45–58, 1993)     

The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana

Summary RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, ³J_NH and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel -sheets and three -helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 -protons located in the loop-1 region, an 2 helix, and three -strands (1, 3 and 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.Abbreviations DQF-COSY double-quantum-filtered correlation spectroscopy - DTT dithiothreitol - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - PE-1 N-terminal pyroglutamate - RC-RNase ribonuclease from the oocyte of Rana catesbeiana - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-trimethylsilylpropionate-2,2,3,3-d ₄     

Evolution of Amino Acid Propensities under Stability-Mediated Epistasis

Site-specific amino acid preferences are influenced by the genetic background of the protein. The preferences for resident amino acids are expected to, on average, increase over time because of replacements at other sites—a nonadaptive phenomenon referred to as the “evolutionary Stokes shift.” Alternatively, decreases in resident amino acid propensity have recently been viewed as evidence of adaptations to external environmental changes. Using population genetics theory and thermodynamic stability constraints, we show that nonadaptive evolution can lead to both positive and negative shifts in propensities following the fixation of an amino acid, emphasizing that the detection of negative shifts is not conclusive evidence of adaptation. By examining propensity shifts from when an amino acid is first accepted at a site until it is subsequently replaced, we find that ≈50% of sites show a decrease in the propensity for the newly resident amino acid while the remaining sites show an increase. Furthermore, the distributions of the magnitudes of positive and negative shifts were comparable. Preferences were often conserved via a significant negative autocorrelation in propensity changes—increases in propensities often followed by decreases, and vice versa. Lastly, we explore the underlying mechanisms that lead propensities to fluctuate. We observe that stabilizing replacements increase the mutational tolerance at a site and in doing so decrease the propensity for the resident amino acid. In contrast, destabilizing substitutions result in more rugged fitness landscapes that tend to favor the resident amino acid. In summary, our results characterize propensity trajectories under nonadaptive stability-constrained evolution against which evidence of adaptations should be calibrated.     

Possible chromosomal location of genes determining the osmoregulation of wheat

Summary Stress-induced free amino acid accumulation in the presence of 0.7 M mannitol has been compared in tissue cultures of moderately stress-tolerant Chinese Spring and stress-sensitive Cappelle Desprez cultivars and in disomic chromosome substitution lines of Cappelle Desprez into Chinese Spring. The profile of amino acid accumulation was different in the two parents. The amino acid concentration of the substitution lines belonging to the A, B and D genomes, respectively, altered characteristically under stress condition. The Cappelle Desprez chromosomes associated with non-ionic osmotic stress-induced free amino acid accumulation were 5A and 5D.     

Differential coupling efficiency of chemically activated amino acid to tRNA

Summary Interaction based on possible chemical affinity of an amino acid for tRNA was examined as a model for the aminoacylation of primitive tRNA without aid of an enzyme system. Two types of reaction were carried out and compared. One was the acyl linkage of amino acid to the 5-terminal phosphate of a tRNA activated as an imidazolide. The other was the incorporation of an amino acid activated as an imidazolide into 2(3)-hydroxyl groups of intact tRNA. Both types of reaction indicated that none of the amino acids tested had any selectivity for the tRNAs examined. However, the rates of reaction with a given tRNA were different among amino acids. In the second type of reaction, amino acids were found mainly at loop-out regions of tRNA, but not at either its 5- or 3-terminal sitesOneA ₂₆₀ unit is defined as an amount of material which gives an absorption of 1.0 at 260 nm when dissolved in 1 ml water and measured with a 1-cm light path