Number Of Amino Acid Differences Research Articles

New insights into the criteria of functional heterozygosity of the Apis mellifera complementary sex determining gene-Discovery of a functional allele pair differing by a single amino acid.

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations.

Estimating antigenic distances between GII.4 human norovirus strains

Human norovirus (HuNoV) is an etiological agent of acute gastroenteritis. Viral protein 1 (VP1) is the major capsid protein and the determinant of antigenicity in HuNoV. GII.4 has been the dominant VP1-genotype in most of HuNoV seasons. Here an attempt was made to estimate the magnitude of antigenic difference, defined as the antigenic distance, between GII.4 HuNoV strains from the comparison of amino acid sequences for VP1. Antigenic distance was estimated more accurately assuming an exponential decline in the amount of increase in the antigenic distance along with an increment in the number of amino acid differences than assuming a constant increase. Although antigenic sites A, C, D, E, and G appeared to be the major determinant of antigenicity, estimation accuracy was sometimes improved by adding other regions. These results suggested that it may be suitable to construct a model based on the entire region of VP1 for estimating antigenic distances between GII.4 HuNoV strains.

Molecular‐level HLA mismatch is associated with rejection and worsened graft survival in heart transplant recipients – a retrospective study

The aim was to evaluate the association of molecular‐level human leukocyte antigen (HLA) mismatching with post‐transplant graft survival, rejection, and cardiac allograft vasculopathy (CAV). We retrospectively analyzed all primary cardiac transplant recipients between 01/1984‐06/2016. 1167 patients fulfilled inclusion criteria and had HLA typing information available. In 312 donor‐recipient pairs, typing at serological split antigen level was available. We used the Epitope MisMatch Algorithm to calculate the number of amino acid differences in antibody‐verified HLA eplets (amino acid mismatch load (AAMM)) between donor and recipient. Patients with a higher HLA‐DR AAMM load had inferior 1‐year graft survival (hazard ratio [HR], 1.14; 95% confidence interval [CI], 1.01–1.28). The HLA‐AB AAMM load showed no impact on graft survival. In the subgroup with available split‐level information, we observed an inferior graft survival for a higher HLA‐DR AAMM load 3 months after transplantation (HR, 1.22; 95% CI, 1.04–1.44) and a higher risk for rejection for an increasing HLA‐AB (HR, 1.70; 95% CI, 1.29–2.24) and HLA‐DR (HR, 1.32; 95% CI, 1.09–1.61) AAMM load. No impact on the development of CAV was found. Molecular‐level HLA mismatch analysis could serve as a tool for risk stratification after heart transplantation and might take us one step further into precision medicine.

Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro.

Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires 14C-labeled substrate and allows to determine its incorporation into phytoene as well as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase homologs from various plant species.

Differences in the amino acid composition of the antigen epitopes of the VP7 protein of Russian rotaviruses with the G9 genotype and the vaccine strains RotaTeq, Rotavac, and Rotarix

Introduction. Rotaviruses of group A (RVA) with genotype G9P[8] are a common cause of acute gastroenteritis in children in Russia. In Nizhny Novgorod, the part of G9P[8] among all RVA strains reached 63.1% during 20162017 epidemic season. Two live rotavirus vaccines, RotaTeq and Rotarix have been successfully introduced into the national immunization programs worldwide. In addition, the Indian vaccine Rotavac, based on the strain with G9P[8] genotype, is used on a regional level. The parent strains for all mentioned vaccines were isolated more than 30 years ago. There is no data about phylogenetic analysis and comparative analysis of antigenic epitopes of Russian G9P[8] wild-type isolates and vaccine strains. In the present study, for the first time, we provide a comparative phylogenetic analysis and research of the amino acid composition of the B- and T-cell epitopes of the VP7 protein between Russian rotaviruses with the G9 genotype and the vaccine strains in RotaTeq, Rotarix and Rotavac composition. Materials and methods. The nucleotide and amino acid sequences of the VP7 gene of RVA with genotype G9 were studied. The rotaviruses had been previously isolated from children hospitalized with acute gastroenteritis in the infectious hospital in Nizhny Novgorod during 20112016. Results. A phylogenetic analysis of the nucleotide sequences of the VP7 gene showed that the Nizhny Novgorod strains belong to the G9-III allele. Based on the amino acid sequences VP7, three B-cell epitopes (71a, 71b and 72) and two T-cell epitopes (1628 aa and 4052 aa) were analyzed. The smallest number of substitutions was found in the RotaTeq vaccine registered in Russia: from 0 to 3 aa differences at the epitope. The same (from 0 to 3 aa differences at the epitope) was found between the wild-type strains RVA and the Rotavac vaccine. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G9 strains (from 3 to 10 aa at the epitope). Conclusion. In the present work, based on nucleotide sequences VP7 gene, we provide phylogenetic and comparative analyses of the amino acid composition of antigenic epitopes of G9 RVA isolated in Russia vs rotavirus strains in vaccines RotaTeq, Rotavac and Rotarix. The accumulation of mutations in antigenic epitopes can help the virus to escape the immune response. Continuous molecular monitoring of wild-type RVA strains is necessary for estimation of the possible impact of vaccines on the genotype diversity of the rotavirus population in the wild and to monitor the emergence of novel antigenic variants.

Few Amino Acid Exchanges Expand the Substrate Spectrum of Monocarboxylate Transporter 10.

Monocarboxylate transporters (MCTs) belong to the SLC16 family within the major facilitator superfamily of transmembrane transporters. MCT8 is a thyroid hormone transporter mutated in the Allan-Herndon-Dudley syndrome, a severe psychomotor retardation syndrome. MCT10 is closely related to MCT8 and is known as T-type amino acid transporter. Both transporters mediate T3 transport, but although MCT8 also transports rT3 and T4, these compounds are not efficiently transported by MCT10, which, in contrast, transports aromatic amino acids. Based on the 58% amino acid identity within the transmembrane regions among MCT8 and MCT10, we reasoned that substrate specificity may be primarily determined by a small number of amino acid differences between MCT8 and MCT10 along the substrate translocation channel. Inspecting the homology model of MCT8 and a structure-guided alignment between both proteins, we selected 8 amino acid positions and prepared chimeric MCT10 proteins with selected amino acids changed to the corresponding amino acids in MCT8. The MCT10 mutant harboring 8 amino acid substitutions was stably expressed in Madin-Darby canine kidney 1 cells and found to exhibit T4 transport activity. We then successively reduced the number of amino acid substitutions and eventually identified a minimal set of 2-3 amino acid exchanges which were sufficient to allow T4 transport. The resulting MCT10 chimeras exhibited KM values for T4 similar to MCT8 but transported T4 at a slower rate. The acquisition of T4 transport by MCT10 was associated with complete loss of the capacity to transport Phe, when Tyr184 was mutated to Phe.

US-like isolates of porcine epidemic diarrhea virus from Japanese outbreaks between 2013 and 2014

Since late 2013, outbreaks of porcine epidemic diarrhea virus (PEDV) have reemerged in Japan. In the present study, we observed a high detection rate of PEDV, with 72.5 % (148/204) of diarrhea samples (suckling, weaned, and sows) and 88.5 % (77/87) of farms experiencing acute diarrhea found to be positive for PEDV by reverse transcription PCR. Sequencing and phylogenic analyses of the partial spike gene and ORF3 of PEDV demonstrated that all prevailing Japanese PEDV isolates belonged to novel genotypes that differed from previously reported strains and the two PEDV vaccine strains currently being used in Japan. Sequence and phylogenetic analysis revealed prevailing PEDV isolates in Japan had the greatest genetic similarity to US isolates and were not vaccine-related. Unlike vaccine strains, all prevailing field PEDV isolates in Japan were found to have a number of amino acid differences in the neutralizing epitope domain, COE, which may affect antigenicity and vaccine efficacy. The present study indicates recent PEDV isolates may have been introduced into Japan from overseas and highlights the urgent requirement of novel vaccines for controlling PEDV outbreaks in Japan.

Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BackgroundThe calcium‐binding 2EF‐hand protein Phl p 7 from timothy grass pollen is a highly cross‐reactive pollen pan‐allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7‐specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen‐specific immunotherapy (SIT).MethodsWe studied epitope specificity, cross‐reactivity, affinity and cross‐protection of mAb102.1F10 towards homologous calcium‐binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross‐reactivity of patients' IgE and mAb102.1F10 to calcium‐binding allergens and peptides thereof were studied with quantitative RAST‐based methods, in ELISA, basophil activation and IgE‐facilitated allergen presentation experiments.ResultsAllergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross‐reacted with allergic patients' IgE. mAb102.1F10 bound the C‐terminal portion of Phl p 7 in a calcium‐dependent manner. It cross‐reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross‐inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross‐reactivity.ConclusionsOur results demonstrate that a small number of amino acid differences among cross‐reactive allergens can reduce the affinity of binding by a SIT‐induced IgG and thus limit cross‐protection.

Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines

Two live, attenuated rotavirus A (RVA) vaccines, Rotarix and RotaTeq, have been successfully introduced into national immunization programs worldwide. The parent strains of both vaccines were obtained more than 30 years ago. Nonetheless, only very limited data are available on the molecular similarity of the vaccine strains and their genetic relationships to the wild-type strains circulating within the territory of Russian Federation. In this study, we have determined the nucleotide sequences of the genes encoding the viral proteins VP7 and VP4 (the globular domain VP8*) of vaccine strains and natural isolates of rotaviruses in Nizhny Novgorod, Russia. The VP7 and VP4 proteins contain antigenic sites that are the main targets of neutralizing antibodies. Phylogenetic analysis based on VP4 and VP7 showed that the majority of the natural RVA isolates from Nizhny Novgorod and the vaccine strains belong to different clusters. Four amino acids within the VP7 antigenic sites were common in both the wild-type and vaccine strains. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G2 strains (19 residues out of 29). From 3 to 5 amino acid differences per strain were identified in the antigenic sites of VP4 (domain VP8*) between wild-type strains and the vaccine RotaTeq, and 6-8 substitutions were found when they were compared with the vaccine strain Rotarix. For the first time, immunodominant T-cell epitopes of VP7 were analyzed, and differences in the sequences between the vaccine and the wild-type strains were found. The accumulation of amino acid substitutions in the VP7 and VP4 antigenic sites may potentially reduce the immune protection of vaccinated children from wild-type strains of rotavirus.

Genome editing a mouse locus encoding a variant histone, H3.3B, to report on its expression in live animals

Chromatin remodeling via incorporation of histone variants plays a key role in the regulation of embryonic development. The histone variant H3.3 has been associated with a number of early events including formation of the paternal pronucleus upon fertilization. The small number of amino acid differences between H3.3 and its canonical counterparts (H3.1 and H3.2) has limited studies of the developmental significance of H3.3 deposition into chromatin due to difficulties in distinguishing the H3 isoforms. To this end, we used zinc-finger nuclease (ZFN) mediated gene editing to introduce a small C-terminal hemagglutinin (HA) tag to the endogenous H3.3B locus in mouse embryonic stem cells (ESCs), along with an internal ribosome entry site (IRES) and a separately translated fluorescent reporter of expression. This system will allow detection of expression driven by the reporter in cells, animals, and embryos, and will facilitate investigation of differential roles of paternal and maternal H3.3 protein during embryogenesis that would not be possible using variant-specific antibodies. Further, the ability to monitor endogenous H3.3 protein in various cell lineages will enhance our understanding of the dynamics of this histone variant over the course of development.

The Hidden Benefit of CREG Matching.

Matching for crossreactive groups (CREGs) of HLA antigens has been proposed to increase equity in the distribution of deceased donor organs among racial groups and to improve graft survival. Mixed results have been reported for the effect on graft survival; however, CREG matching may reduce the extent of sensitization following graft failure. We examined sera from 704 patients who were shown to be sensitized after renal transplantation and assessed the frequencies of antibodies to mismatches at the HLA-A, -B, -DR, and -DQ loci. Sera were tested using multiplexed bead assays with purified HLA antigen targets. Patients were categorized by whether or not a mismatched antigen was a CREG match. At each locus, the frequency of an antibody to a mismatched antigen was significantly less when the mismatch was a CREG match than when it was not (A: 38 v. 62%, P=0.000004; B: 33 v. 55%, P=0.00004; DR: 40 v. 65%, P=0.0003; DQ: 46 v 71%, P=0.02). However, there was great variability when mismatches were considered by individual specificity with the extent of reduction in antibody frequency ranging from 0 to 72% for different crossreactive antigens. Also, the impact of various CREG matches within a crossreactive group was highly variable and was, in some cases, directional. E.g., there was a 53% reduction in frequency of antibody to B8 when the patient had a B7 in their phenotype but no reduction in antibody frequency in the converse situation, i.e., when the patient was a B8 and the mismatch was a B7. In contrast to the protective effect of B7 is that a B7 mismatch when the patient does not have a B8 in their phenotype, results in antibody to B8 with nearly the same frequency as does a mismatch for B8. There are programs that calculate the number of amino acid differences between two alleles but CREG matching can be done manually and without the need for typing at the allele level. Since most transplants will have HLA mismatches, selecting donors with mismatches that are crossreactive with the patient's own antigens (i.e. CREG mismatches) may be important for patients, such as pediatric patients, who are likely to need retransplantation. However, the variability in the impact of CREG mismatches on sensitization must be considered in the donor selection process.

Genetic variability of human metapneumo‐ and bocaviruses in children with respiratory tract infections

ObjectivesThe genotypic analysis of human metapneumo-(HMPV) and boca-(HBoV) viruses circulating in Greece and their comparison to reference and other clinical strains.DesignGenetic analysis of representative strains over three consecutive winter seasons of the years 2005–2008.SettingRepresentative positive specimens for HMPV and HBoV from paediatric patients of healthcare units and hospitals in Southern Greece with influenza-like illness or other respiratory tract infections.SampleSeven to ten positive specimens for either HMPV or HBoV from each winter period. In total, 24 specimens positive for HMPV and 26 for HBoV, respectively.Main outcome measuresSequence diversity of HMPV and HBoV strains by sequencing the complete G and VP1/VP2 genes, respectively.ResultsIn total, 24 HMPV strains were found to have a 92–100% nucleotide and a 85.9–100% amino acid identity. Phylogenetic analysis based on the number of amino acid differences, revealed circulation of 4 different subclusters belonging to genetic lineage B2. Similarly, analysis of 26 HBoV strains indicated that 22 clustered within genotype St2, 2 into genotype St1 and the remaining 2 formed a third cluster derived from potential recombination between different St1 genotype strains. St2 HBoV genotype was observed throughout the whole observation period whereas St1 only during the second and the third winter period. Higher levels of heterogeneity were observed between HMPV compared to HBoV strains.ConclusionsPhylogenetic analysis revealed circulation of one single lineage (B2) for HMPV viruses and predominance of St2 genotype for HBoV viruses. A possible recombination between St1 genotype strains of HBoV was observed.

Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera

Deciphering the evolutionary processes driving nucleotide variation in multiallelic genes is limited by the number of genetic systems in which such genes occur. The complementary sex determiner (csd) gene in the honey bee Apis mellifera is an informative example for studying allelic diversity and the underlying evolutionary forces in a well-described model of balancing selection. Acting as the primary signal of sex determination, diploid individuals heterozygous for csd develop into females, whereas csd homozygotes are diploid males that have zero fitness. Examining 77 of the functional heterozygous csd allele pairs, we established a combinatorical criteria that provide insights into the minimum number of amino acid differences among those pairs. Given a data set of 244 csd sequences, we show that the total number of csd alleles found in A. mellifera ranges from 53 (locally) to 87 (worldwide), which is much higher than was previously reported (20). Using a coupon-collector model, we extrapolate the presence of in total 116–145 csd alleles worldwide. The hypervariable region (HVR) is of particular importance in determining csd allele specificity, and we provide for this region evidence of high evolutionary rate for length differences exceeding those of microsatellites. The proportion of amino acids driven by positive selection and the rate of nonsynonymous substitutions in the HVR-flanking regions reach values close to 1 but differ with respect to the HVR length. Using a model of csd coalescence, we identified the high originating rate of csd specificities as a major evolutionary force, leading to an origin of a novel csd allele every 400,000 years. The csd polymorphism frequencies in natural populations indicate an excess of new mutations, whereas signs of ancestral transspecies polymorphism can still be detected. This study provides a comprehensive view of the enormous diversity and the evolutionary forces shaping a multiallelic gene.

Suboptimal protection against H5N1 highly pathogenic avian influenza viruses from Vietnam in ducks vaccinated with commercial poultry vaccines

Domestic ducks are the second most abundant poultry species in many Asian countries including Vietnam, and play a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI) [FAO]. In this study, we examined the protective efficacy in ducks of two commercial H5N1 vaccines widely used in Vietnam; Re-1 containing A/goose/Guangdong/1/1996 hemagglutinin (HA) clade 0 antigens, and Re-5 containing A/duck/Anhui/1/2006 HA clade 2.3.4 antigens. Ducks received two doses of either vaccine at 7 and at 14 or 21 days of age followed by challenge at 30 days of age with viruses belonging to the HA clades 1.1, 2.3.4.3, 2.3.2.1.A and 2.3.2.1.B isolated between 2008 and 2011 in Vietnam. Ducks vaccinated with the Re-1 vaccine were protected after infection with the two H5N1 HPAI viruses isolated in 2008 (HA clades 1.1 and 2.3.4.3) showing no mortality and limited virus shedding. The Re-1 and Re-5 vaccines conferred 90–100% protection against mortality after challenge with the 2010 H5N1 HPAI viruses (HA clade 2.3.2.1.A); but vaccinated ducks shed virus for more than 7 days after challenge. Similarly, the Re-1 and Re-5 vaccines only showed partial protection against the 2011 H5N1 HPAI viruses (HA clade 2.3.2.1.A and 2.3.2.1.B), with a high proportion of vaccinated ducks shedding virus for more than 10 days. Furthermore, 50% mortality was observed in ducks vaccinated with Re-1 and challenged with the 2.3.2.1.B virus. The HA proteins of the 2011 challenge viruses had the greatest number of amino acid differences from the two vaccines as compared to the viruses from 2008 and 2009, which correlates with the lesser protection observed with these viruses. These studies demonstrate the suboptimal protection conferred by the Re-1 and Re-5 commercial vaccines in ducks against H5N1 HPAI clade 2.3.2.1 viruses, and underscore the importance of monitoring vaccine efficacy in the control of H5N1 HPAI in ducks.

Promoter-Specific Transcription Inhibition in Staphylococcus aureus by a Phage Protein

Phage G1 gp67 is a 23 kDa protein that binds to the Staphylococcus aureus (Sau) RNA polymerase (RNAP) σ(A) subunit and blocks cell growth by inhibiting transcription. We show that gp67 has little to no effect on transcription from most promoters but is a potent inhibitor of ribosomal RNA transcription. A 2.0-Å-resolution crystal structure of the complex between gp67 and Sau σ(A) domain 4 (σ(A)(4)) explains how gp67 joins the RNAP promoter complex through σ(A)(4) without significantly affecting σ(A)(4) function. Our results indicate that gp67 forms a complex with RNAP at most, if not all, σ(A)-dependent promoters, but selectively inhibits promoters that depend on an interaction between upstream DNA and the RNAP α-subunit C-terminal domain (αCTD). Thus, we reveal a promoter-specific transcription inhibition mechanism by which gp67 interacts with the RNAP promoter complex through one subunit (σ(A)), and selectively affects the function of another subunit (αCTD) depending on promoter usage.

A Proposed Algorithm Predictive for Cytotoxic T Cell Alloreactivity

Previously, we showed that with an increasing number of amino acid differences in single HLA class I-mismatched molecules, the probability of T cell alloreactivity decreases. It is unlikely that every amino acid difference will affect T cell alloreactivity in a similar way; we hypothesized that the effect of an amino acid difference may be dependent on its position and/or physicochemical properties. We selected 131 patient/donor pairs with either a single HLA-A or -C mismatch in the graft-versus-host direction and that were compatible for HLA-B, -DRB1, and -DQB1. The alloreactive CTL precursor (CTLp) frequency was determined and associated with the amino acid differences between the single HLA class I mismatches. In the β sheet, only amino acids that are noncompatible in their physicochemical properties affect T cell alloreactivity, whereas in the α helices, both compatible and noncompatible amino acids affect CTLp outcome. Positions 62, 63, 73, 76, 77, 80, 99, 116, 138, 144, 147, and 163 were bivariately associated with CTLp outcome, irrespective of the total number of amino acid differences. In multivariate analysis, positions 62, 63, 73, 80, 116, 138, 144, and 163 were found to be most predictive for negative CTLp outcome. These results formed the basis for a weighted predictive mismatch score; pairs with the highest mismatch scores are estimated to be 13 times more likely to have a negative CTLp. This new algorithm may be a tool in donor selection for hematopoietic stem cell transplantation.

Females prefer good genes: MHC-associated mate choice in wild and captive tuco-tucos

The major histocompatibility complex (MHC) genes are one of the most suitable candidates for elucidating the genetic bases of mate choice in vertebrates, given the potential benefits in terms of immunocompetence that can be passed to the offspring through MHC-associated mate choice. Female mate choice may favour males that possess particular MHC alleles or those with diverse MHC genotypes (good genes hypothesis), or males that possess MHC genotypes that differ from that of the female (compatibility hypothesis). Our goal was to evaluate mate choice in relation to MHC genotype in the subterranean rodent Ctenomys talarum. Using both laboratory and field analyses, we investigated whether the (1) number of shared MHC alleles between males and females, (2) number of amino acid differences between female and male MHC alleles, (3) MHC heterozygosity of males, (4) number of amino acid differences between male MHC alleles, and (5) frequency of MHC alleles of males differ between preferred and nonpreferred males in the laboratory and between potential sires and random males from the population in the field. In the laboratory, our results indicate that MHC alleles of preferred males differ in fewer amino acids compared to MHC alleles of nonpreferred males. Concomitantly, in the field, MHC alleles of possible sires differed in fewer amino acids than those of random males in the population. Plus, possible sires were more heterozygous and carried distinct MHC alleles compared with random males, thus providing more support to the ‘good genes’ hypothesis. We discuss the possible reasons why MHC-based mate choice was more evident in the field and the implications of such a female mating strategy in the subterranean environment.

Genetic Analyses Reveal Differences in the VP7 and VP4 Antigenic Epitopes between Human Rotaviruses Circulating in Belgium and Rotaviruses in Rotarix and RotaTeq

Two live-attenuated rotavirus group A (RVA) vaccines, Rotarix (G1P[8]) and RotaTeq (G1-G4, P[8]), have been successfully introduced in many countries worldwide, including Belgium. The parental RVA strains used to generate the vaccines were isolated more than 20 years ago in France (G4 parental strain in RotaTeq) and the United States (all other parental strains). At present, little is known about the relationship between currently circulating human RVAs and the vaccine strains. In this study, we determined sequences for the VP7 and VP4 outer capsid proteins of representative G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] RVAs circulating in Belgium during 2007 to 2009. The analyses showed that multiple amino acid differences existed between the VP7 and VP4 antigenic epitopes of the vaccine viruses and the Belgian isolates, regardless of their G and P genotypes. However, the highest variability was observed among the circulating G1P[8] RVA strains and the G1 and P[8] components of both RVA vaccines. In particular, RVA strains of the P[8] lineage 4 (OP354-like) showed a significant number of amino acid differences with the P[8] VP4 of both vaccines. In addition, the circulating Belgian G3 RVA strains were found to possibly possess an extra N-linked glycosylation site compared to the G3 RVA vaccine strain of RotaTeq. These results indicate that the antigenic epitopes of RVA strains contained in the vaccines differ substantially from those of the currently circulating RVA strains in Belgium. Over time, these differences might result in selection for strains that escape the RVA neutralizing-antibody pressure induced by vaccines.

Relationships between rust resistance genes at the M locus in flax.

Genes at the M locus in flax (Linum usitatissimum) that confer resistance to flax rust (Melampsora lini) occur in complex haplotypes containing up to 15 related genes or gene fragments. We have cloned two additional functional resistance genes at this locus, M1 and M3, by transposon tagging and candidate gene approaches, and investigated the genetic relationships between four genes (M, M1, M3 and M4) by recombination analysis. M1 and M3, like M, are members of the nucleotide binding site, leucine-rich repeat (NBS-LRR) family. Comparisons of the predicted M1 and M3 amino acid sequences with M and L6 reveal that: (i) M1 contains four additional LRRs, probably as a result of an unequal crossover event between duplicated regions; (ii) M1 shares large segments of exact identity with M and M3, indicative of intragenic recombination events; and (iii) a large number of amino acid differences are scattered throughout the M, M1 and M3 proteins. Recombination analysis (here and in previous studies) has revealed that M readily recombines with M1, M3 and M4, whereas these three genes fail to recombine despite large family sizes (>5800) in two test-cross families, suggesting that they may occupy allelic positions in the gene cluster. Several restriction fragment length polymorphism markers within or near the M locus were mapped with respect to seven crossover events between M and M1. The results of this and previous studies provide evidence of structural differences between: (i) homoeologous loci in the different genomes of flax; (ii) different haplotypes at the M locus; (iii) different resistance genes in the M group; and (iv) the flanking regions downstream of M locus resistance genes.

Variation in Homeodomain DNA Binding Revealed by High-Resolution Analysis of Sequence Preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.