Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy- terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.     

Saturating Mutagenesis of an Essential Gene: a Majority of the Neisseria gonorrhoeae Major Outer Membrane Porin (PorB) Is Mutable

The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin''s multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.     

The Impact of Message Mutation on the Fitness of a Genetic Code

The Standard Genetic Code is organized such that similar codons encode similar amino acids. One explanation suggested that the Standard Code is the result of natural selection to reduce the fitness ``load' that derives from the mutation and mistranslation of protein-coding genes. We review the arguments against the mutational load-minimizing hypothesis and argue that they need to be reassessed. We review recent analyses of the organization of the Standard Code and conclude that under cautious interpretation they support the mutational load-minimizing hypothesis. We then present a deterministic asexual model with which we study the mode of selection for load minimization. In this model, individual fitness is determined by a protein phenotype resulting from the translation of a mutable set of protein-coding genes. We show that an equilibrium fitness may be associated with a population with the same genetic code and that genetic codes that assign similar codons to similar amino acids have a higher fitness. We also show that the number of mutant codons in each individual at equilibrium, which determines the strength of selection for load minimization, reflects a long-term evolutionary balance between mutations in messages and selection on proteins, rather than the number of mutations that occur in a single generation, as has been assumed by previous authors. We thereby establish that selection for mutational load minimization acts at the level of an individual in a single generation. We conclude with comments on the shortcomings and advantages of load minimization over other hypotheses for the origin of the Standard Code. Received: 4 April 2001 / Accepted: 22 October 2001     

The question of natural selection against mutable-to-terminator codons

Codons that differ from a terminator triplet by only a single base are sometimes thought, because of their greater risk of undergoing a harmful mutation, to be kept at a reduced frequency by natural selection. The present study on human genes shows that these codons are infrequent, but their low frequency is not directly related to their risk of mutation to terminator. Rather, it is a consequence of their ending in A or G; comparable A-ending and G-ending codons that are not mutable to terminator are also infrequent. Natural selection does not appear to have depressed the frequency of mutable-to-terminator codons by directly eliminating some of them individually. It may have depressed their frequency indirectly by adjusting mutation rates in the population so that most of the purine-ending codons--and consequently most of the mutable-to-terminator ones as well--are infrequent.     

Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes

At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence.     

Compositional correlation and codon usage studies in Buchnera aphidicola

Compositional distributions in three different codon positions as well as codon usage biases of all available DNA sequences of Buchnera aphidicola genome have been analyzed. It was observed that GC levels among the three codon positions is I>II>III as observed in other extremely high AT rich organisms. B. aphidicola being an AT rich organism is expected to have A and/or T at the third positions of codons. Overall codon usage analyses indicate that A and/or T ending codons are predominant in this organism and some particular amino acids are abundant in the coding region of genes. However, multivariate statistical analysis indicates two major trends in the codon usage variation among the genes; one being strongly correlated with the GC contents at the third synonymous positions of codons, and the other being associated with the expression level of genes. Moreover, codon usage biases of the highly expressed genes are almost identical with the overall codon usage biases of all the genes of this organism. These observations suggest that mutational bias is the main factor in determining the codon usage variation among the genes in B. aphidicola.     

Deletions in the large (beta) subunit of a hetero-oligomeric aminoacyl-tRNA synthetase

Glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an alpha 2 beta 2 quaternary structure. The two subunits are encoded by a single mRNA with the region for alpha (303 codons) subunit followed by that for beta (689 codons) subunit. Five COOH-terminal deletions in the beta subunit coding region have been created. Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro. This has been done in the presence of free alpha subunit and, additionally, with alpha subunit that is fused by its carboxyl terminus to the amino terminus of each of the beta subunit deletion proteins. With a fused or unfused alpha chain, over 100 amino acids can be deleted from the carboxyl terminus of the beta chain without loss of in vivo complementation of a delta glyS (deletion) strain. Further analysis shows that the alpha subunit and approximately the amino-terminal half of the beta subunit are sufficient for the adenylate synthesis activity. In particular, a deletion of 306 amino acids from the COOH terminus of the beta subunit has little effect on the Km parameter for ATP or glycine in the pyrophosphate exchange reaction. The tRNA-dependent step in aminoacylation requires additional beta subunit sequences on the COOH-terminal side of those needed for adenylate synthesis. In these respects, the functional organization of the beta chain parallels that of several aminoacyl-tRNA synthetases which have only homologous subunits. In the case of the glycine enzyme, however, the heterologous alpha subunit is required for the elucidation of activities encoded by functional determinants of the beta chain.     

Mouse haemoglobin beta chains. Comparative sequence data on adult major and minor beta chains from two species, Mus musculus and Mus cervicolor.

In inbred Mus musculus several different beta chains are known. In certain strains two beta chains are produced in unequal amounts by the two closely linked genes of the doublet breeding unit allele Hbb(d): Betadmaj and betadmin. One strain has a variant doublet allele, Hbb(p), which produces a variant minor beta chain, betapmin (the major beta chain, betapmaj, may not differ from betadmaj chain). Certain other strains have a singlet allele, Hbb(s), that produces only one beta chain, betas. Other species have different beta-chain patterns. In M. cervicolor two variant major beta chains are found, betacmaj (d-like) and betacmaj (s-like), both of which were found associated with minor beta chains. M. caroli has only one, 'Leporelike' beta chain, with structural features characteristic predominantly of betadmin chain in the N-terminal half and of betadmaj chain in the C-terminal half. The present paper presents sequence data on betas, betadmaj, betadmin, betapmin and betacmaj (d-like) chains. The data on betadmin chain cover almost the whole of that chain and show a minimum of nine differences from betadmaj chain and two from betapmin chain. It is suggested that the data on the beta chains of the various species show evidence for the past occurrence of double crossovers over regions within a gene coding for only one or a few amino acids, which events can be explained by the 'hybrid DNA' models of genetic recombination. Supplementary information on the amino acid sequence of the proteins has been deposited as Supplementary Publication SUP 50067 (36 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.     

Identification of B beta chain domains involved in human fibrinogen assembly.

Fibrinogen chains are assembled in a stepwise manner in the rough endoplasmic reticulum prior to secretion of the final six-chain dimeric molecule. Previous studies indicated that the synthesis of B beta may be a rate-limiting factor in the assembly of human fibrinogen. To determine the domains of B beta which interact with the other two component chains of fibrinogen, deletion mutants of B beta were transiently co-expressed, together with A alpha and gamma chains, in COS cells, and fibrinogen assembly and secretion were measured. Deletion of the COOH-terminal half of the B beta chain (amino acids 208-461) did not affect assembly and secretion. Assembly of A alpha, gamma, and B beta also occurred when the first NH2-terminal 72 amino acids of B beta were deleted, but not when 93 amino acids were deleted. This indicates that the B beta domain between amino acids 73 and 93 is necessary for the assembly of the three fibrinogen chains. This domain marks the start of the alpha-helical "coiled-coil" region of fibrinogen.     

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.     

A general model of codon bias due to GC mutational bias

Background

In spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns.

Principal Findings

In analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively.

Conclusions

The model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.     

Construction of genetic code from evolutionary stability

The construction of the genetic code is investigated based on a stability principle. The concept and formulation of mutational deterioration (MD) of the genetic code is proposed. It is proved that the degeneracies of codon multiplets obey the rule to best resist MD. The MD for each ideal multiplet of codons is expressed by four parameters and it takes on a minimum value for real distributions of codons in the multiplet. Then the global mutational deterioration (GMD) of code table is calculated and the minimal code is deduced. The domain-like distribution of hydrophobic and hydrophilic amino acids on the genetic code is explained from the minimization of GMD. It is demonstrated that the standard code is approximately GMD-minimal. By introducing some constraints that are related to the initial condition of the system, we have deduced the standard genetic code from the minimization of GMD. The minimization shows the general trend of evolutionary process to some stable state while the constraints reflect a 'frozen accident.' Many deviant codon assignments are also explained through MD minimization assuming the changeable degrees of degeneracies for some multiplets. So, a possible answer to the question of "Why are synonymous codons and amino acids distributed in the code table just as they are?" is given.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Sequence similarities of protein kinase peptide substrates and inhibitors: comparison of their primary structures with immunoglobulin repeats

Forty original sequences of peptide substrates and inhibitors of protein kinases and phosphatases were aligned in a chain matrix without artificial gaps. Fifteen protein kinase peptide substrates and inhibitors (PKSI peptides) contained a common dipeptide ArgArg and also additional important tetra-, tri- and dipeptide homologies. Three further peptide substrates were significantly similar to these peptides but lacked the ArgArg dipeptide. Sequence comparison of individual PKSI peptides revealed probabilistically restricted consensus sequence—PKSI motif—comprising 8 homologous and 13 non-randomly distributed amino acids without considering mutation analysis. This template motif was compared with the consensus sequences of 12 different immunoglobulin domains. In 11 of 12 these domains, the starts of homologous segments were found at nearly the same domain related sites, beginning with serine. A single-triplet mutation, of any of the first two triplet bases that encode equally localized amino acids in each of the two sequence sets (PKSI and Ig) revealed additional homologies with the other set. A primary derived motif version composed of 9 homologous and seven non-randomly distributed amino acids was consequently established by its feedback projection into the original sequence sets. This procedure yielded a second preliminary motif version (revised motif) formed by a sequence of 9 homologous amino acids and two non-randomly distributed amino acids. In addition, three shorter oligopeptide motifs called important stereotypes were derived, based on repeated homology between Ig chains and the revised motif. The most extensive similarities in terms of these stereotypes occurred in the CH2 and CH4 domains of Ig peptides, and inhibitors of cAMP dependent protein kinase and protein kinase A. Further comparisons based on a reference sequence set arranged with the aid of feedback projection revealed a lower similarity between variable Ig chains reflected in a decreased number of homologous amino acids. Two final motif versions, FMC and FMV, were found in two different subsets of constant and variable Ig chains, respectively. FMC was composed of seven homologous and one non-randomly distributed amino acids forming the dispersed structure STLR(C)LVSD, whereas 6 homologous and one questionable amino acid constituted FMV. Only CH4 and CH1 domain segments contained all five high-incidence amino acids, which represented a higher level of similarity than homologous amino acids of all preliminary and final motifs. Four such amino acids were present also in three PKSI peptides. All similarities described here occur in domain segments positionally overlapping with the CDR1 region of variable chains. The results are discussed in terms of immunoglobulin evolution, the position of Fc receptor binding sites and degeneration or mutability of the triplets of motif-constituting amino acids.     

Chemical structure of two fragments of human serum albumin and their location in the albumin molecule.

1. 'Inhibitor fragment' isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.     

Conformation biases of amino acids based on tripeptide microenvironment from PDB database

We have constructed a bank (FTTP) of tendentious factors of three states of three-peptide units from PDB database based on conformational dihedral angle library and demonstrated that amino acid biases toward protein secondary structure are present in natural protein sequences. Our research results reveal that 20 standard amino acids fall into three groups: nine residues inclined to alpha-helix with a common character (e.g. direct side chain aliphatic residues or positive/negative charged residues) arrange in three grades, viz EA, QKRLD, and MN, in turn; seven residues are apt to beta-strand with 2'-branched side chain aliphatic residues or benzyl-included residues, namely PV, IYTC, and F, in three ranks; and four residues SHWG show a double tendency to both alpha and beta. Noticeably, proline has the strongest ability to form extended conformation, especially the Re value up to 9.5298 at position 3 (Table 3). Thus, biases of codons show an evident tendency in protein folding, where GC-rich codons are mainly in charge of forming contracted conformation, especially the codon's first letter plays a dominant role in translating the genomic GC signature into protein sequences and structures. So, biases of amino acids will play an important role in protein folding, folding codons, refining domain, structure prediction, and structural genomics/proteomics.     

On the information content of the genetic code

In living organisms 20 amino acids along with the terminator value(s) are encoded by 64 codons giving a degeneracy of the codons as described by the genetic code. A basic theoretical problem of genetic codes is to explain the particular distribution of degeneracies of partitions involved in the codes. In this work the degeneracy problem is considered in the framework of information theory. It is shown by direct numerical evaluation of a certain degeneracy information function associated with the genetic code that the degeneracy of the codes is observed to be related to the optimization of this function.