Evolutionary processes and evolutionary noise at the molecular level

Summary On account, notably, of a competition between different component functions for individual sites in polypeptide chains, each protein molecule represents a functional compromise, with some functions optimized, but the overall state of the molecule –suboptimal–. The proposal is made that the selection coefficient relating to a protein molecule under given conditions can in principle be broken down into partial selection coefficients relevant to the different functions that the molecule carries out. At generalfunction sites, each fixation improves some function, while others deteriorate, at first nonsignificantly, and the overall adaptive state of the molecule fluctuates around its maximum. A selective mechanism is described whereby kaleidoscopic changes in primary structure at variable sites are indefinitely promoted, independently of any environmental changes and with the molecule remaining close to a state of maximal overall adaptation. The paradoxical aspect of this proposal is analyzed. The implication of specific functions in substitutions at general-function sites is noted. Further, it is shown that a certain category of changes in the internal environment of the organism can be integrated into the constantenvironment model for selection. Genetic sufficiency is considered a notion more adequate than genetic optimality for describing biological fitness and for providing a basis for the present model. On this basis selection occurs without genetic load. Multipolymorphism is one of the consequences. Several lines of evidence, in particular observations on polymorphism in deep sea organisms, seem to support the model. It is pointed out that it provides a theoretical foundation for a molecular evolutionary clock. The theoretical constancy of the clock depends on the constancy of functional density. The question of the evolution of functional density is examined. Comparisons of observed substitution frequencies with values expected on a random basis are rejected as a measure of the contribution to evolution of nondetermination. They are considered to reflect a hierarchy in the resistance of the molecules to different amino acid residues as substituents. A limited component of –true– randomness, again accompanied by selection, is on the other hand provided by the model. Most amino acid substitutions are considered evolutionary noise, even though noise compatible with selection. It is proposed that evolutionarily significant substitutions may be identified by monitoring changes in functional density and weighted functional density.Directeur de Recherche at Centre National de la Recherche Scientifique, Paris.     

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets,Sodium Channels

To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na_vs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na_vs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na_vs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na_vs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na_vs.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.     

Reduction of synonymous substitutions in the core protein gene of hepatitis C virus

Molecular evolutionary analyses were carried out to elucidate the phylogenetic relationships, the evolutionary rate, and the divergence times of hepatitis C viruses. Using the nucleotide sequences of the viruses isolated from various locations in the world, we constructed phylogenetic trees. The trees showed that strains isolated from a single location were not necessarily clustered as a group. This suggests that the viruses may be transferred with blood on a worldwide scale. We estimated the evolutionary rates at synonymous and nonsynonymous sites for all genes in the viral genome. We then found that the rate (1.35 × 10^–3 per site per year) at synonymous sites for the C gene was much smaller than those for the other genes (e.g., 6.29 × 10^–3 per site per year for the E gene). This indicates that a special type of functional constraint on synonymous substitutions may exist in the C gene. Because we found an open reading frame (ORF) with the C gene region, the possibility exists that synonymous substitutions for the C gene are constrained by the overlapping ORF whose reading frame is different from that of the C gene. Applying the evolutionary rates to the trees, we also suggest that major groups of hepatitis C viruses diverged from their common ancestor several hundred years ago. Correspondence to: T. Gojobori     

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.     

Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest major roles for this structural property in shaping the differences of evolutionary adaptability in the three routes.     

Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.     

Divergent Regulation of Muscarinic Binding Sites and Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

Summary The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [³H]N-methyl-4-piperidyl benzilate ([⁴H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, withK _d values of 1.2 ± 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 ± 0.1 at 14 weeks up to 0.7 ± 0.4 pmol/mg protein at 24 weeks.The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [³H]acetylcholine/min/mg protein. These were mostly true acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10^–5 M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10^–5 M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similarK _m values toward [³H]acetylcholine (ca. 4 × 10^–4 M ^–1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 ± 50 and 19 ± 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.I.B. took part in this work as partial fulfillment of the requirements of the Sackler Faculty of Medicine for an M.D. degree.     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca²⁺ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca²⁺ channels and their adaptation to sperm biology through metazoan evolution.     

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Hans Leemhuis acknowledges financial support from the Netherlands Organization for Scientific Research (NWO).     

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum (Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.     

Protein co-evolution, co-adaptation and interactions

Co-evolution has an important function in the evolution of species and it is clearly manifested in certain scenarios such as host–parasite and predator–prey interactions, symbiosis and mutualism. The extrapolation of the concepts and methodologies developed for the study of species co-evolution at the molecular level has prompted the development of a variety of computational methods able to predict protein interactions through the characteristics of co-evolution. Particularly successful have been those methods that predict interactions at the genomic level based on the detection of pairs of protein families with similar evolutionary histories (similarity of phylogenetic trees: mirrortree). Future advances in this field will require a better understanding of the molecular basis of the co-evolution of protein families. Thus, it will be important to decipher the molecular mechanisms underlying the similarity observed in phylogenetic trees of interacting proteins, distinguishing direct specific molecular interactions from other general functional constraints. In particular, it will be important to separate the effects of physical interactions within protein complexes (‘co-adaptation') from other forces that, in a less specific way, can also create general patterns of co-evolution.     

Specificity of mRNA Folding and Its Association with Evolutionarily Adaptive mRNA Secondary Structures

The secondary structure is a fundamental feature of both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs). However, our understanding of the secondary structures of mRNAs, especially those of the coding regions, remains elusive, likely due to translation and the lack of RNA-binding proteins that sustain the consensus structure like those binding to ncRNAs. Indeed, mRNAs have recently been found to adopt diverse alternative structures, but the overall functional significance remains untested. We hereby approach this problem by estimating the folding specificity, i.e., the probability that a fragment of an mRNA folds back to the same partner once refolded. We show that the folding specificity of mRNAs is lower than that of ncRNAs and exhibits moderate evolutionary conservation. Notably, we find that specific rather than alternative folding is likely evolutionarily adaptive since specific folding is frequently associated with functionally important genes or sites within a gene. Additional analysis in combination with ribosome density suggests the ability to modulate ribosome movement as one potential functional advantage provided by specific folding. Our findings reveal a novel facet of the RNA structurome with important functional and evolutionary implications and indicate a potential method for distinguishing the mRNA secondary structures maintained by natural selection from molecular noise.     

Tracing Primordial Protein Evolution through Structurally Guided Stepwise Segment Elongation

The understanding of how primordial proteins emerged has been a fundamental and longstanding issue in biology and biochemistry. For a better understanding of primordial protein evolution, we synthesized an artificial protein on the basis of an evolutionary hypothesis, segment-based elongation starting from an autonomously foldable short peptide. A 10-residue protein, chignolin, the smallest foldable polypeptide ever reported, was used as a structural support to facilitate higher structural organization and gain-of-function in the development of an artificial protein. Repetitive cycles of segment elongation and subsequent phage display selection successfully produced a 25-residue protein, termed AF.2A1, with nanomolar affinity against the Fc region of immunoglobulin G. AF.2A1 shows exquisite molecular recognition ability such that it can distinguish conformational differences of the same molecule. The structure determined by NMR measurements demonstrated that AF.2A1 forms a globular protein-like conformation with the chignolin-derived β-hairpin and a tryptophan-mediated hydrophobic core. Using sequence analysis and a mutation study, we discovered that the structural organization and gain-of-function emerged from the vicinity of the chignolin segment, revealing that the structural support served as the core in both structural and functional development. Here, we propose an evolutionary model for primordial proteins in which a foldable segment serves as the evolving core to facilitate structural and functional evolution. This study provides insights into primordial protein evolution and also presents a novel methodology for designing small sized proteins useful for industrial and pharmaceutical applications.     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

Fifty years after Enlow and Brown's Comparative histological study of fossil and recent bone tissues (1956–1958): A review of Professor Donald H. Enlow's contribution to palaeohistology and comparative histology of bone

Fifty years after the publication of Enlow and Brown's seminal paper (1956–1958), this historical analysis of Professor Donald H. Enlow's works emphasizes their influence on the evolution of palaeohistology, comparative bone histology, and bone biology in general. Comparative analysis of recent and fossil bone tissues has shown the great variability of bone at the tissue level (histodiversity). Historically, Enlow's works have greatly influenced a shift in the reinterpretation of the causes of bone histodiversity, from phylogenetic to more functional (ontogenetic) explanations. This allows us to consider the issue of complex causalities in evolutionary biology.     

The herpes simplex virus DNA polymerase: Analysis of the functional domains

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597–961, comprising the homologous sequence regions IV–IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3′-to-5′ exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 ± 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3′-to-5′ exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072–1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.     

The analysis of hidden electrophoretic variation: Interspecific electrophoretic differentiation and amino acid divergence

Summary In a study of 25 human variants and 23 evolutionary alleles of hemoglobin we show that intraspecific and interspecific patterns of electrophoretic variability are not comparable. Significant deviation from the predicted electrophoretic differentiation between evolutionary alleles is normally found only when amino acid sequence divergence exceeds 10%. When two sequences had diverged at less than 30 out of 287 amino acid residues sites, only 7% of comparisons showed significant deviations from the expected difference of electrophoretic mobility, while significant deviation was shown by 57% of comparisons involving 30–40 residue differences, by 79% in the case of 51–60 differences and by all of the comparisons involving more than 60 differences. In contrast, human variants, which differ by only one or two amino acid residues (less than 1% difference), had significant deviations in 58% of comparisons. Those mutations that appear as fixed differences in the evolutionary material probably represent only a subset of the mutations which can appear within the species. The results suggest that statistical comparisons such as genetic distance may not measure the same process within a species as between species. This is due not to inherent problems with the statistic, but rather to inherent differences in the nature of molecular changes that are detectable by electrophoresis at different stages of population divergence.