Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.     

Characterization of the nicotinic acetylcholine receptor subunit gene Mdalpha2 from the house fly, Musca domestica

A nicotinic acetylcholine receptor (nAChR) subunit gene, Mdalpha2, was isolated and characterized from the house fly, Musca domestica. This is the first nAChR family member cloned from house flies. Mdalpha2 had a cDNA of 2,607 bp, which included a 696 bp 5'-untranslated region (UTR), an open reading frame of 1,692 bp, and a 219 bp 3'-UTR. Its deduced amino acid sequence possesses the typical characteristics of nAChRs. Mdalpha2 genomic sequence was 11.2 kb in length in the aabys strain and 10.9 kb in the OCR strain, including eight exons and seven introns. Based on the deduced amino acid sequence, Mdalpha2 had the closest phylogenetic relationship to the Drosophila melanogaster Dalpha2 and Anopheles gambiae Agamalpha2, and a similar genomic structure to Dalpha2. Quantitative real-time PCR analysis showed that Mdalpha2 is expressed in the head and the thorax at 150- and 8.5-fold higher levels than in the abdomen. Linkage analysis of a Mdalpha2 polymorphism indicates this gene is on autosome 2. The importance of these results in understanding the diversity and phylogenetic relationships of insect nAChRs, the physiology of nAChRs in the house fly, and the utility of nAChR sequences in resistance detection/monitoring is discussed.     

Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission

The neuromuscular junction (NMJ) is a complex structure that efficiently communicates the electrical impulse from the motor neuron to the skeletal muscle to induce muscle contraction. Genetic and autoimmune disorders known to compromise neuromuscular transmission are providing further insights into the complexities of NMJ function. Congenital myasthenic syndromes (CMSs) are a genetically and phenotypically heterogeneous group of rare hereditary disorders affecting neuromuscular transmission. The understanding of the molecular basis of the different types of CMSs has evolved rapidly in recent years. Mutations were first identified in the subunits of the nicotinic acetylcholine receptor (AChR), but now mutations in ten different genes - encoding post-, pre- or synaptic proteins - are known to cause CMSs. Pathogenic mechanisms leading to an impaired neuromuscular transmission modify AChRs or endplate structure or lead to decreased acetylcholine synthesis and release. However, the genetic background of many CMS forms is still unresolved. A precise molecular classification of CMS type is of paramount importance for the diagnosis, counselling and therapy of a patient, as different drugs may be beneficial or deleterious depending on the molecular background of the particular CMS.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high‐level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.     

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100_209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the _mutgp100_201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the _mutgp100_209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8⁺ T cell stimulation in vitro similar to the _wtgp100_209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8⁺ T cell response also towards N-terminally extended versions of the minimal epitope.     

Infestation of arboreal nests of coatis by triatomine species,vectors of Trypanosoma cruzi,in a large Neotropical wetland

The coati (Nasua nasua, Carnivora) is a medium‐sized mammal common in the Pantanal of Brazil. Unlike most mammals, coatis construct arboreal nests used for resting and reproduction. In this region, the coati is an important host of Trypanosoma cruzi, the causative agent of Chagas disease. There are two possible routes through coatis can be infected by T. cruzi: the oral route or the vectorial route. However, the relative importance of each of these routes in the infection of coatis and its role in the sylvatic cycle of the parasite are unknown. Our objectives were to investigate: (i) whether coati nests were infested by triatomine bugs, (ii) what species were frequent in the nests, (iii) whether the triatomines in nests were infected by T. cruzi, and (iv) what were the food resources of these triatomines. Eight of the 24 nests sampled were infested with triatomines, a total of 37 specimens of at least two species (Rhodnius stali and Triatoma sordida). In one nest, R. stali and T. sordida co‐occurred and both fed on multiple resources, including coatis. This is the first report of triatomines occurring in arboreal nests of coatis. The co‐occurrence of two different genera of triatomine vectors and coatis within the limited space of the coati nests provide multiple opportunities for the exchange of the protozoan parasite through both the vectorial and oral transmission routes.