Impact of bias discrepancy and amino acid usage on estimates of the effective number of codons used in a gene, and a test for selection on codon usage

The effective number of codons (Nc) used in a gene is one of the most commonly used measures of synonymous codon usage bias, owing much of its popularity to the fact that it is species independent and that simulation studies have shown that it is less dependent of gene length than other measures. In this paper I provide a clear and practically meaningful definition of bias discrepancy (BD; when the degree of codon bias varies within a degeneracy class). Moreover I evaluate the impact of BD and amino acid usage on estimates of Nc. It is shown that both factors have a significant effect on accuracy and precision. Both amino acid usage and BD influence accuracy considerably, especially in short genes. Finally, I demonstrate how the definition of bias discrepancy can be applied to investigate if codon usage is influenced by selection and I discuss this test in relation to the incongruous literature that exists for Buchnera sp. APS and Borrelia burgdorferi.     

Towards a resolution on the inherent methodological weakness of the "effective number of codons used by a gene"

Recently Anders Fuglsang provided a modified way for calculating N(c) when biased discrepancy is present in a gene [Biochem. Biophys. Res. Commun. 317 (2004) 957]. Instead of taking the average codon homozygosity for each synonymous family type (as proposed by Wright) [Gene 87 (1990) 23] Fuglsang considered codon homozygosity of each amino acid individually. Marsashi and Najafabadi [Biochem. Biophys. Res. Commun. 324 (2004) 1] in their recent article demonstrated that the readjustment for overestimation at the level of individual amino acids results in loss of considerable amount of information. Immediately after the publication of Marsashi and Najafabadi, Fuglsang proposed that codon homozygosities can be calculated based on the classical population genetics [Biochem. Biophys. Res. Commun. 327 (2005) 1]. Though Fuglsang's approach is a novel one, it fails when any of the amino acids are absent in a gene. However, the inherent cause of overestimation at the level of individual amino acids is still obscured in the literature. Here in this communication we have presented a general condition where effective number of codons is overestimated using Wright's formula and also we propose a new way to calculate N(c), which is independent of amino acid composition.     

Estimating the effective number of breeders from heterozygote excess in progeny

The heterozygote-excess method is a recently published method for estimating the effective population size (Ne). It is based on the following principle: When the effective number of breeders (Neb) in a population is small, the allele frequencies will (by chance) be different in males and females, which causes an excess of heterozygotes in the progeny with respect to Hardy-Weinberg equilibrium expectations. We evaluate the accuracy and precision of the heterozygote-excess method using empirical and simulated data sets from polygamous, polygynous, and monogamous mating systems and by using realistic sample sizes of individuals (15-120) and loci (5-30) with varying levels of polymorphism. The method gave nearly unbiased estimates of Neb under all three mating systems. However, the confidence intervals on the point estimates of Neb were sufficiently small (and hence the heterozygote-excess method useful) only in polygamous and polygynous populations that were produced by <10 effective breeders, unless samples included > approximately 60 individuals and 20 multiallelic loci.     

Efficient expression of gene variants that harbour AGA codons next to the initiation codon

In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNA^Arg4 as well as a short 5′-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNA^Arg4.     

Genetics leads the way to the accomplices of presenilins

Matrix metalloproteinases are enzymes that regulate tissue behavior by interactions with extracellular matrix proteins. RECK, a membrane-anchored inhibitor of MMPs was recently characterized for its role in development, tissue homeostasis, and for tumor angiogenesis.     

Estimating effective population size from samples of sequences: inefficiency of pairwise and segregating sites as compared to phylogenetic estimates.

It is known that under neutral mutation at a known mutation rate a sample of nucleotide sequences, within which there is assumed to be no recombination, allows estimation of the effective size of an isolated population. This paper investigates the case of very long sequences, where each pair of sequences allows a precise estimate of the divergence time of those two gene copies. The average divergence time of all pairs of copies estimates twice the effective population number and an estimate can also be derived from the number of segregating sites. One can alternatively estimate the genealogy of the copies. This paper shows how a maximum likelihood estimate of the effective population number can be derived from such a genealogical tree. The pairwise and the segregating sites estimates are shown to be much less efficient than this maximum likelihood estimate, and this is verified by computer simulation. The result implies that there is much to gain by explicitly taking the tree structure of these genealogies into account.     

Nb_HetEx: a program to estimate the effective number of breeders

The program "Nb_HetEx" estimates the effective number of breeders (N(b)) that produced the sampled progeny based on genotype counts contained in that sample. When the number of breeders is very small, there is an excess of heterozygotes in their progeny: the smaller the number of breeders, the larger the heterozygote excess. The Nb_HetEx program also estimates N(e) through the temporal method.     

Using genomic inbreeding coefficient estimates for homozygosity mapping of rare recessive traits: application to Taybi-Linder syndrome

The use of inbred patients whose exact genealogy may not be available is of primary interest in mapping genes involved in rare recessive diseases. We show here that this can be achieved by estimating inbreeding coefficients from the patients' genomic information and using these estimates to perform homozygosity mapping. We show the interest of the approach by mapping a gene for Taybi-Linder syndrome to chromosome 2q, with the use of a key patient with no genealogical information.     

Amino acid and codon usage profiles: adaptive changes in the frequency of amino acids and codons

In the work presented, the changes in codon and amino acid contents have been studied as a function of environmental conditions by comparing pairs of homologs in a group of extremophilic/non-extremophilic genomes. Our results obtained based on such analysis highlights a number of notable observations: (i) the overall preference of amino acid usages in the proteins of a given organism is significantly affected by major environmental factors. The changes in amino acid preferences (amino acid usage profiles) in an extremophile compared to its non-extremophile relative recurs in the organisms of similar extreme habitats. (ii) On the other hand, changes in codon usage preferences in these extremophilic/non-extremophilic pairs, lack such persistency not only in different genome-pairs but also in the individual genes of a specific pair. (iii) We have noted a correlation between cellular function and codon usage profiles of the genes in the studied pairs. (iv) Based on this correlation, we could obtain a decent prediction of cellular functions solely based on codon usage profile data. (v) Comparisons made between two sets of randomly generated genomes suggest that different patterns of codon usage changes in genes of different functional categories result in a partial resistance towards the changes in the concentration of a given amino acid. This buffering capacity might explain the observed differences in codon usage trends in genes of different functions. In the end, we suggest codon usage and amino acid profiles as powerful tools that can be utilized to improve function predictions and genome-environment mappings.     

Multilevel selection 2: Estimating the genetic parameters determining inheritance and response to selection

Interactions among individuals are universal, both in animals and in plants and in natural as well as domestic populations. Understanding the consequences of these interactions for the evolution of populations by either natural or artificial selection requires knowledge of the heritable components underlying them. Here we present statistical methodology to estimate the genetic parameters determining response to multilevel selection of traits affected by interactions among individuals in general populations. We apply these methods to obtain estimates of genetic parameters for survival days in a population of layer chickens with high mortality due to pecking behavior. We find that heritable variation is threefold greater than that obtained from classical analyses, meaning that two-thirds of the full heritable variation is hidden to classical analysis due to social interactions. As a consequence, predicted responses to multilevel selection applied to this population are threefold greater than classical predictions. This work, combined with the quantitative genetic theory for response to multilevel selection presented in an accompanying article in this issue, enables the design of selection programs to effectively reduce competitive interactions in livestock and plants and the prediction of the effects of social interactions on evolution in natural populations undergoing multilevel selection.     

Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes

BACKGROUND: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. METHODS: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. RESULTS: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. CONCLUSIONS: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.     

Potential bias in inbreeding depression estimates when using pedigree relationships to assess the degree of homozygosity for loci under selection

A potential bias in estimation of inbreeding depression when using pedigree relationships to assess the degree of homozygosity for loci under selection is indicated. A comparison of inbreeding coefficients based on either pedigree or genotypic frequencies indicated that, as a result of selection, the inbreeding coefficient based on pedigree might not correspond with the random drift of allelic frequencies. Apparent differences in average levels of both inbreeding coefficients were obtained depending on the genetic model (additive versus dominance, initial allelic frequencies, heritability) and the selection system assumed (no versus mass selection). In the absence of selection, allelic frequencies within a small population change over generations due to random drift, and the pedigree-based inbreeding coefficient gives a proper assessment of the accompanying probability of increased homozygosity within a replicate by indicating the variance of allelic frequencies over replicates. With selection, in addition to random drift, directional change in allelic frequencies is not accounted for by the pedigree-based inbreeding coefficient. This result implies that estimation of inbreeding depression for traits under either direct or indirect selection, estimated by a regression of performance on pedigree-based coefficients, should be carefully interpreted.Deceased     

Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.     

Multiple paternity: determining the minimum number of sires of a large brood

We describe an efficient algorithm for determining exactly the minimum number of sires consistent with the multi-locus genotypes of a mother and her progeny. We consider cases where a simple exhaustive search through all possible sets of sires is impossible in practice because it would take too long to complete. Our algorithm for solving this combinatorial optimization problem avoids visiting large parts of search space that would not result in a solution with fewer sires. This improvement is of particular importance when the number of allelic types in the progeny array is large and when the minimum number of sires is expected to be large. Precisely in such cases, it is important to know the minimum number of sires: this number gives an exact bound on the most likely number of sires estimated by a random search algorithm in a parameter region where it may be difficult to determine whether it has converged. We apply our algorithm to data from the marine snail, Littorina saxatilis.     

The "cellular state": the way to regain specificity and diversity in hormone action

In contrast with the high specificity achieved in the effects of hormones and growth factors by their interaction with a large number of membrane receptors, a loss of information seems to take place due to the small number of second messenger systems. To retain specificity one has to consider the cellular state as defined in a state machine by a pattern of activity. Different molecular mechanisms are considered as possible candidates to establish such states following the initial ligand-receptor interaction and the activation of one or several second messengers.