Primary structure of the variable regions of two canine immunoglobulin heavy chains.

The complete amino acid sequences of the variable regions of two canine immunoglobulin heavy chains have been determined by automated Edman degradation and found to be strongly homologous to the human VHIII subgroup. The canine sequences were identical with each other at 76 of 113 residue positions. Twenty-three of the 37 differences are located within the four hypervariable regions previously defined by the sequences of several human VHIII proteins. Forty-five of 77 framework residue positions are invariant in the seven human and two canine VHIII proteins which have been completely sequences. The canine proteins are 78% homologous to the framework of the human prototype. Phylogenetically associated residues before the first hypervariable region were confirmed and several potential phylogenetically associated residues were identified between the first and third hypervariable regions. This study represents the first complete amino acid sequences of VH regions of spontaneously occurring, nonhuman homogeneous immunoglobulins. The date demonstrate a high degree of preservation of VHIII structure in another species.     

Positive selection of digestive Cys proteases in herbivorous Coleoptera

Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within –or close to– amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins.     

Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.     

Immunoglobulin heavy chains from anti-inulin myeloma proteins: evidence for a new heavy chain joining segment

Immunoglobulin heavy chains have been shown to be encoded by at least 3 widely separated genetic elements, designated variable (V), diversity (D), and joining (J), which undergo rearrangement during somatic differentiation to produce the active gene form. The D segment codes for a portion of the 3rd hypervariable region and thus potentially contributes significantly to structural diversity in this portion of the molecule. Heavy chains from anti-inulin proteins are unusual in that they essentially lack a 3rd hypervariable region. Thus, if a D segment exists in these proteins, it is extremely short, possibly 1 to 2 amino acids, and more likely serves a framework function rather than introduces structural diversity in the 3rd hypervariable region. We have completed the heavy chain variable region amino acid sequence from proteins AMPC1 and T957 bringing to 6 the number of complete sequences from this group. All of these proteins lack a 3rd hypervariable region. In addition, substitutions are found within the J segments of AMPC1 and T957, which are unlikely to be generated by the recombination event. The occurrence of Pro at position 105 in both of these J segments in contrast to the Gln found in all other heavy chains using this J segment suggests the possible existence of a previously unidentified J segment gene.     

A Molecular Genetic Approach to Understanding the Evolution of Immunoglobulin Gene Structure and Diversity

Immune function in higher vertebrates is mediated primarilyby multimeric glycoproteins found in the serum and on the surfacesof lymphoid cells. These molecules possess common structuralfeatures suggesting that they belong to a supergene family whichmay have originated from a common ancestral gene. Some multigenicmembers of the supergene family undergo unique forms of chromosomalrearrangement during somatic development. We have identifiedimmunoglobulin heavy chainvariable region (V_H) homologs in speciesrepresenting critical points in the vertebrate radiation, examinedtheir nucleotide sequences and found high degrees of organizationalhomology as well as localized regions of extended nucleotide(and amino acid) sequence identity with mammalian V_H genes.The unexpected high degree of nucleotide sequence identity suggeststhat within this multigene family, selection may be operatingat both the DNA and polypeptide levels.Using several differentapproaches, the V_H gene families in lower vertebrates have beenshown to be remarkably complex, discounting the possibilitythat a reduced number of germline genes accounts for the apparentlyrestricted natureof lower vertebrate immune responses. The lowervertebrate germline V_H genes possess prototypic recombinationsignal sequences, implicated in the somatic reorganization ofmammalian immunoglobulin variable region genes, and segmentalreorganizationresembling that seen in mammals has been observed in an elasmobranch.The detection of a recombination element flanked by short, directrepeats within the intervening sequence of one reptilian V_Hgene suggests that these sequences may be mobile, perhaps functioningoutside of the immunoglobulin loci in other developmental processes.The complex nature of the variable region gene families andtheir capacity to undergo structural change during somatic developmentsuggest that unique genetic mechanisms may govern their evolutionarystabilization and diversification.     

Canonical structures for the hypervariable regions of immunoglobulins

We have analysed the atomic structures of Fab and VL fragments of immunoglobulins to determine the relationship between their amino acid sequences and the three-dimensional structures of their antigen binding sites. We identify the relatively few residues that, through their packing, hydrogen bonding or the ability to assume unusual phi, psi or omega conformations, are primarily responsible for the main-chain conformations of the hypervariable regions. These residues are found to occur at sites within the hypervariable regions and in the conserved beta-sheet framework. Examination of the sequences of immunoglobulins of unknown structure shows that many have hypervariable regions that are similar in size to one of the known structures and contain identical residues at the sites responsible for the observed conformation. This implies that these hypervariable regions have conformations close to those in the known structures. For five of the hypervariable regions, the repertoire of conformations appears to be limited to a relatively small number of discrete structural classes. We call the commonly occurring main-chain conformations of the hypervariable regions "canonical structures". The accuracy of the analysis is being tested and refined by the prediction of immunoglobulin structures prior to their experimental determination.     

Relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity in the mouse

The relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity were studied by comparing germline gene sequences with their rearranged counterparts for the mouse VH, V kappa, and V lambda genes. The mutation rate at the amino acid level was estimated to be 7.0% in the first and second complementarity- determining regions (CDRs) and 2.0% in the framework regions (FRs). The difference in the mutation rate at the nucleotide level between the CDRs and FRs was of the same order of magnitude as that for the amino acid level. Analysis of amino acid diversity or nucleotide diversity indicated that the contribution of somatic mutation to immunoglobulin diversity is approximately 5%. However, the contribution of somatic mutation to the number of different amino acid sequences of immunoglobulins is much larger than that estimated by the analysis of amino acid diversity, and more than 90% of the different immunoglobulins seem to be generated by somatic mutation. Examination of the pattern of nucleotide substitution has suggested that clonal selection after somatic mutation may not be as strong as generally believed.      

Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey.

The entire phosphoprotein (P) and nucleocapsid (N) protein gene sequences and deduced amino acid sequences for 18 selected vesicular stomatitis virus isolates representative of the natural genetic diversity within the New Jersey serotype are reported. Phylogenetic analysis of the data using maximum parsimony allowed construction of evolutionary trees for the individual genes and the combined N, P, and glycoprotein (G) genes of these viruses. Virtually identical rates of nucleotide substitutions were found for each gene, indicating that evolution of these genes occurs at essentially the same rate. Although up to 19 and 17% sequence differences were evident in the P and N genes, respectively, no variation in gene length or evidence of recombinational rearrangements was found. However, striking evolutionary differences were observed among the amino acid sequences of vesicular stomatitis virus New Jersey N, P, and G proteins. The N protein amino acid sequence was the most highly conserved among the different isolates, indicating strong functional and structural constraints. Conversely, the P protein amino acid sequences were highly variable, indicating considerably fewer constraints or greater evolutionary pressure on the P protein. Much of the remarkable amino acid variability of the P protein resided in a hypervariable domain located between amino acids 153 and 205. The variability within this region would be consistent with it playing a structural role as a spacer to maintain correct conformational presentation of the separate active domains of this multifunctional protein. In marked contrast, the adjacent domain I of the P protein (previously thought to be under little evolutionary constraint) contained a highly conserved region. The colocalization of a short, potentially functional overlapping open reading frame to this region may explain this apparent anomaly.     

Evolution of Ig DNA sequence to target specific base positions within codons for somatic hypermutation

Ig variable (V) region genes are subjected to a somatic hypermutation process as B lymphocytes participate in immune reactions to protein Ags. Although little is known regarding the mechanism of mutagenesis, a consistent hierarchy of trinucleotide target preferences is evident. Analysis of trinucleotide regional distributions predicted and we now empirically confirm the surprising finding that the framework 2 region of kappa V region genes is highly mutable despite its importance to the structural integrity and function of the Ab molecule. Interestingly, much of this mutability appears to be focused on the third codon position where synonymous substitutions are most likely to occur. We also observed a trend for high predicted mutability for codon positions 1 and 2 in complementarity-determining regions. Consequently, amino acid replacements should occur at a higher rate in complementarity-determining regions than in framework regions due to the distribution and subsequent targeting of microsequences by the mutation mechanism. Our results reveal a subtle tier of V region gene evolution in which DNA sequence has been molded to direct mutations to specific base positions within codons in a manner that minimizes damage and maximizes the benefits of the somatic hypermutation process.     

An evolutionarily conserved network of amino acids mediates gating in voltage-dependent potassium channels

A novel sequence-analysis technique for detecting correlated amino acid positions in intermediate-size protein families (50-100 sequences) was developed, and applied to study voltage-dependent gating of potassium channels. Most contemporary methods for detecting amino acid correlations within proteins use very large sets of data, typically comprising hundreds or thousands of evolutionarily related sequences, to overcome the relatively low signal-to-noise ratio in the analysis of co-variations between pairs of amino acid positions. Such methods are impractical for voltage-gated potassium (Kv) channels and for many other protein families that have not yet been sequenced to that extent. Here, we used a phylogenetic reconstruction of paralogous Kv channels to follow the evolutionary history of every pair of amino acid positions within this family, thus increasing detection accuracy of correlated amino acids relative to contemporary methods. In addition, we used a bootstrapping procedure to eliminate correlations that were statistically insignificant. These and other measures allowed us to increase the method's sensitivity, and opened the way to reliable identification of correlated positions even in intermediate-size protein families. Principal-component analysis applied to the set of correlated amino acid positions in Kv channels detected a network of inter-correlated residues, a large fraction of which were identified as gating-sensitive upon mutation. Mapping the network of correlated residues onto the 3D structure of the Kv channel from Aeropyrum pernix disclosed correlations between residues in the voltage-sensor paddle and the pore region, including regions that are involved in the gating transition. We discuss these findings with respect to the evolutionary constraints acting on the channel's various domains. The software is available on our website     

Haplotypic Divergence Coupled with Lack of Diversity at the Arabidopsis Thaliana Alcohol Dehydrogenase Locus: Roles for Both Balancing and Directional Selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as ``hypervariable'''' on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana''s tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.     

Human profilin. Molecular cloning, sequence comparison, and chromosomal analysis

Profilin is an ubiquitous 12-15-kDa actin monomer-binding protein, the amino acid sequence of which was previously reported for the cow and Acanthamoeba. In the latter species, two isoforms of profilin have been identified. We have isolated full-length profilin cDNA clones from a human HepG2 library. All clones have the same nucleotide sequence, and Northern blot and RNase protection analyses of human tissues indicate that all tissues have the same approximately 850 base message, and provide no evidence of alternative message splicing. This result strongly implies a single profilin isoform in human cells, although differential post-translational modifications have not been excluded. Northern blot analysis extends the tissue distribution of profilin to include epithelial, muscle, and renal tissues. Comparison of the predicted human profilin amino acid sequence with that of published bovine profilin indicates 90% identity with a single 3-residue deletion in the human sequence. Southern blot analysis of somatic cell hybrid DNA indicates at least four dispersed genetic loci in the human genome hybridize with the profilin cDNA as well as untranslated region fragments, suggesting several of these loci represent pseudogenes of recent evolutionary origin. In addition, 5' and 3' untranslated regions are conserved between humans and rodents, implying a functional role for these regions of the profilin gene.     

Mutation matrices and physical-chemical properties: Correlations and implications

To investigate how the properties of individual amino acids result in proteins with particular structures and functions, we have examined the correlations between previously derived structure-dependent mutation rates and changes in various physical-chemical properties of the amino acids such as volume, charge, α-helical and β-sheet propensity, and hydrophobicity. In most cases we found the ΔG of transfer from octanol to water to be the best model for evolutionary constraints, in contrast to the much weaker correlation with the ΔG of transfer from cyclohexane to water, a property found to be highly correlated to changes in stability in site-directed mutagenesis studies. This suggests that natural evolution may follow different rules than those suggested by results obtained in the laboratory. A high degree of conservation of a surface residue's relative hydrophobicity was also observed, a fact that cannot be explained by constraints on protein stability but that may reflect the consequences of the reverse-hydrophobic effect. Local propensity, especially α-helical propensity, is rather poorly conserved during evolution, indicating that non-local interactions dominate protein structure formation. We found that changes in volume were important in specific cases, most significantly in transitions among the hydrophobic residues in buried locations. To demonstrate how these techniques could be used to understand particular protein families, we derived and analyzed mutation matrices for the hypervariable and framework regions of antibody light chain V regions. We found a surprisingly high conservation of hydrophobicity in the hypervariable region, possibly indicating an important role for hydrophobicity in antigen recognition. Proteins 27:336–344, 1997. © 1997 Wiley-Liss, Inc.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.     

Predicted functional RNAs within coding regions constrain evolutionary rates of yeast proteins

Functional RNAs (fRNAs) are being recognized as an important regulatory component in biological processes. Interestingly, recent computational studies suggest that the number and biological significance of functional RNAs within coding regions (coding fRNAs) may have been underestimated. We hypothesized that such coding fRNAs will impose additional constraint on sequence evolution because the DNA primary sequence has to simultaneously code for functional RNA secondary structures on the messenger RNA in addition to the amino acid codons for the protein sequence. To test this prediction, we first utilized computational methods to predict conserved fRNA secondary structures within multiple species alignments of Saccharomyces sensu strico genomes. We predict that as much as 5% of the genes in the yeast genome contain at least one functional RNA secondary structure within their protein-coding region. We then analyzed the impact of coding fRNAs on the evolutionary rate of protein-coding genes because a decrease in evolutionary rate implies constraint due to biological functionality. We found that our predicted coding fRNAs have a significant influence on evolutionary rates (especially at synonymous sites), independent of other functional measures. Thus, coding fRNA may play a role on sequence evolution. Given that coding regions of humans and flies contain many more predicted coding fRNAs than yeast, the impact of coding fRNAs on sequence evolution may be substantial in genomes of higher eukaryotes.     

Unique sequences in region VI of the flagellin gene of Salmonella typhi

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.     

Lateral transfer of a lectin-like antifreeze protein gene in fishes

Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.