Balancing selection in Pattern Recognition Receptor signalling pathways is associated with gene function and pleiotropy in a wild rodent

Pathogen‐mediated balancing selection is commonly considered to play an important role in the maintenance of genetic diversity, in particular in immune genes. However, the factors that may influence which immune genes are the targets of such selection are largely unknown. To address this, here we focus on Pattern Recognition Receptor (PRR) signalling pathways, which play a key role in innate immunity. We used whole‐genome resequencing data from a population of bank voles (Myodes glareolus) to test for associations between balancing selection, pleiotropy and gene function in a set of 123 PRR signalling pathway genes. To investigate the effect of gene function, we compared genes encoding (a) receptors for microbial ligands versus downstream signalling proteins, and (b) receptors recognizing components of microbial cell walls, flagella and capsids versus receptors recognizing features of microbial nucleic acids. Analyses based on the nucleotide diversity of full coding sequences showed that balancing selection primarily targeted receptor genes with a low degree of pleiotropy. Moreover, genes encoding receptors recognizing components of microbial cell walls etc. were more important targets of balancing selection than receptors recognizing nucleic acids. Tests for localized signatures of balancing selection in coding and noncoding sequences showed that such signatures were mostly located in introns, and more evenly distributed among different functional categories of PRR pathway genes. The finding that signatures of balancing selection in full coding sequences primarily occur in receptor genes, in particular those encoding receptors for components of microbial cell walls etc., is consistent with the idea that coevolution between hosts and pathogens is an important cause of balancing selection on immune genes.     

Couplings of character and of chirality in the origin of the genetic system

Data from the literature and new data presented here suggest that the genetic system (coding and protein synthesis) is based on relationships of character and structure between amino acids and nucleic acids. Character relationships seem to be anticodonic and structurally the greatest preferences are seen between the heteropair, l-amino acids and d-ribose nucleic acids. However, living systems using the other heteropair must have been equally likely. Homopairing (l-l and d-d) in living systems seems unlikely. Awareness of the heterocoupling of steric forms narrows somewhat the problem of understanding the origin of chirality.     

Antirestriction

Modern data on the molecular mechanisms of antirestriction are reviewed. The systems of inhibition are described for the restriction-modification enzymes of type I in phages and in conjugative plasmids. The phenomenon of alleviation of DNA restriction and its mechanisms is presented for bacteriumEscherichia coli. The principle of protein mimicry of nucleic acids is discussed as a novel way to control biological processes in the cell with reference to antirestriction proteins Ard.     

Generate Western blot protein marker from a single construct

An expression construct, consisting of a tandem arrangement of nucleic acids coding for the constant fragments (Fc) receptor of protein G combined with nucleic acids for the Fc receptor of protein A, was constructed. When the construct was expressed in Escherichia coli, proteins of estimated molecular weights of 25, 30, 50, 58, 80, and 85 kDa were consistently obtained from this expression construct due to possible proteolytic degradation during the cultivation and purification steps. The purified proteins from this single expression construct were used as Western blot protein marker.     

Partition of aminoacyl-tRNA synthetases in two different structural classes dating back to early metabolism: Implications for the origin of the genetic code and the nature of protein sequences

We describe, on the molecular level, a possible fuzzy and primordial translation apparatus capable of synthesizing polypeptides from nucleic acids in a world containing a mixture of coevolving molecules of RNA and proteins already arranged in metabolic cycles (including cofactors). Close attention is paid to template-free systems because they are believed to be the immediate ancestors of this primordial translation apparatus. The two classes of amnoacyl-tRNA synthetases (aaRSs), as seen today, are considered as the remnants of such a simple imprecise translation apparatus and are used as guidelines for the construction of the model. Earlier theoretical work by Bedian on a related system is invoked to show how specificity and stability could have been achieved automatically and rather quickly, starting from such an imprecise system, i.e., how the encoded synthesis of proteins could have appeared. Because of the binary nature of the underlying proto-code, the first genetically encoded proteins would then have been alternating copolymers with a high degree of degeneracy, but not random. Indeed, a clear signal for alternating hydrophobic and hydrophilic residues in present-day protein sequences can be detected. Later evolution of the genetic code would have proceeded along lines already discussed by Crick. However, in the initial stages, the translation apparatus proposed here is in fact very similar to the one postulated by Woese, only here it is given a molecular framework. This hypothesis departs from the paradigm of the RNA world in that it supposes that the origin of the genetic code occurred after the apparition of some functional (statistical) proteins first. Implications for protein design are also discussed.     

Entropies of coding and noncoding sequences of DNA and proteins.

The entropies of protein coding genes from Escherichia coli were calculated according to Boltzmann's formula. Entropies of the coding regions were compared to the entropies of noncoding or miscoding ones. With nucleotides as code units, the entropies of the coding regions, when compared to the entropies of complete sequences (leader and coding region as well as trailer), were seen to be lower but with a marginal statistical significance. With triplets of nucleotides as code units, the entropies of correct reading frames were significantly lower than the entropies of frameshifts +1 and -1. With amino acids as code units, the results were opposite: Biologically functional proteins had significantly higher entropies than proteins translated from the frameshifted sequences. We attempt to explain this paradox with the hypothesis that the genetic code may have the ability of lowering information content (increasing entropy) of proteins while translating them from DNA. This ability might be beneficial to bacteria because it would make the functional proteins more probable (having a higher entropy) than nonfunctional proteins translated from frameshifted sequences.     

Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding

The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.     

Various molecular aspects of cytology and embryology

Our present knowledge of the cell structure, which is largely based on electron microscopy, is compared with what was known a few decades ago, when only light microscopy was available to the cytologist. The importance of cytochemical methods for the detection and localization of macromolecules (nucleic acids, proteins) is stressed. But it is pointed out that further analysis, with biochemical techniques, was required in order to understand the actual mechanisms of macromolecule synthesis in the cell (in particular, the relationships existing between nucleic acids and protein synthesis). The importance of genetical analysis in simple systems such as viruses and bacteria for the development of ‘molecular’ biology is then emphasized: in particular, the work of Avery identifying the ‘transforming principle’ with DNA, of Beadle leading to the ‘one gene, one enzyme’ theory, of the virologists who demonstrated that it is the nucleic acid component of viruses which carries the genetical information, have been of fundamental importance for the development of modern biology. No less important has been the work of the X-ray crystallographers (Crick and Watson, Perutz, Kendrew, etc.) who established the fine structure of nucleic acids and of proteins. A brief review and a schematic representation of present ideas regarding the control exerted by DNA on the synthesis of specific proteins are then given: the main characteristics of the different kinds of RNA's, their interactions for the formation of polysomes, the role of the latter in protein synthesis, the main principles of the genetic codes, are briefly summarized. But cells are, in many respects, more complicated than bacteria. The concepts of molecular biology cannot be applied to cell differentiation without a recognition of the greater complexity of animal and plant cells. They represent, however, a most useful and powerful guide for research in that area: for instance, many aspects of morphogenesis in the unicellular alga Acetabularia and in amphibian eggs can be explained on the assumption that messenger RNA's are produced by the nucleus and stored, in a stabilized form, in the cytoplasm during days or even weeks. This stability of messenger RNA's in eggs and algae is at variance with their short life in bacteria. The behaviour of non-nucleate fragments of Acetabularia is surprising in many respects: they are the site, not only of the synthesis of specific proteins, but even of RNA and DNA net synthesis. Such a synthesis of macromolecules, in the absence of the nucleus is probably linked to the presence of the chloroplasts in this alga: they contain DNA, can synthesize RNA and proteins, and can even increase in number in the absence of the nucleus. The presence of large amounts of DNA in the cytoplasm of many animal eggs raises a number of questions and might account for the extremely important role of the cytoplasm in the very early stages of embryonic development. It is concluded that none of the great problems of cell biology will be solved without the help of the techniques and the theoretical ideas which have been so fruitful for the simpler systems used by the molecular biologists.     

First class transcription termination factors--functional analogs of aminoacyl tRNA

Reviewed and discussed are the recent data demonstrating profound functional similarity between class-1 translation termination factors (RF1 and RF2 in prokaryotes, aRF1 and eRF1 in Archaea and eukaryotes, respectively) and aminoacyl-tRNA as regards their roles in the course of translation on the ribosome. Functional analogy of these two components of the cell protein-synthesizing machinery was suggested long ago; however, numerous experimental proofs have been obtained only recently. This similarity implies that decoding of the genetic information by the ribosomal machine is performed similarly at all stages of translation, though tRNA plays the main role at initiation and elongation, while the protein is most important for termination. Earlier it was found that nucleic acids (ribozymes) can operate like the protein enzymes, and now we have got evidence for the reverse: a protein (translation termination factor) can act like a nucleic acid (tRNA). Thus one can speak of "exchange" of molecular functions between proteins and nucleic acids. Therefore, the profound chemical difference between proteins and nucleic acids is not an insuperable barrier to their mutual functional replacement in certain situations.     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

Informational symmetry breaking between proteins and nucleic acids.

Anti-symmetry of the information-processing mechanisms between proteins and nucleic acids is generalized to informational symmetry breaking between a genetic polymer and an anti-genetic polymer so as not to depend on particular chemical species. In a genetic polymer, e.g. nucleic acids, any sequence can form a closed double-stranded structure with a specific partner sequence. On the other hand, in an anti-genetic polymer, e.g. proteins, a chain could fold to an open multi-stranded structure and reinterpretation of the genetic information through slides or shifts between stacked strands could be induced by external perturbations. The possibility of the informational symmetry breaking by hierarchical organization of a single chemical species, i.e. polypeptides as a genetic polymer and nucleic acids as an anti-genetic polymer, is examined. The informational functions of genetic polymers and anti-genetic polymers in a complex mixture of macromolecules are characterized.     

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.     

In vivo oligomerization of the F conjugative coupling protein TraD

Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.     

Class-1 Translation Termination Factors Are Functional Analogs of Aminoacyl-tRNAs

Reviewed and discussed are the recent data demonstrating profound functional similarity between class-1 translation termination factors (RF1 and RF2 in prokaryotes, aRF1 and eRF1 in Archaea and eukaryotes, respectively) and aminoacyl-tRNA as regards their roles in the course of translation on the ribosome. The functional analogy of these two components of the cell protein-synthesizing machinery was suggested long ago; however, only now it has received ample experimental proof. This similarity implies that decoding of the genetic information by the ribosomal machine is performed likewise at all stages of translation, though tRNA plays the main role at initiation and elongation, while the protein is most important for termination. Earlier it was found that nucleic acids (ribozymes) can operate like the protein enzymes, and now we have got evidence for the reverse: a protein (translation termination factor) can act like a nucleic acid (tRNA). Thus one can speak of exchange of molecular functions between proteins and nucleic acids in specific cases when this provides a biological benefit. Therefore, the profound chemical difference between proteins and nucleic acids is not an insuperable barrier to their functional interchangeability in certain situations.     

Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.     

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5′-NGG-3′ PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5′-NGG-3′ PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.     

Molecular Modelling of Protein-Nucleic Acid Interactions

Abstract

Computer modeling techniques to study the interaction of proteins with nucleic acids are presented. The methods utilize information from genetic and chemical modification experiments and macromolecular structural constraints. These techniques, in addition to computer model building procedures and theoretical energy calculations, are illustrated for the study of the lac and cro repressor-operator systems. Our predicted interactions between lac and its operator agree with those recently reported for lac based upon sequence alignment with the cro repressor. Several molecular models of the putative helical segment of cro interacting with its OR3 operator are presented. These models are reflective of intermediate conformations experienced by the repressor in recognition of the operator sequence. The results of our studies are further discussed in terms of the design of short peptides interacting with nucleic acid sequences and the evolutionary requirements in establishing these repressor interactions.     

Comparison of the acetylation of proteins and nucleic acids

The possibility of acetylation of nucleic acids was examined. Although protein is actively acetylated with [1-(14)C]acetic acid in rat liver systems in vivo and in vitro and in a frog liver system in vivo, nucleic acids are not acetylated under these conditions; nucleic acids purified from these sources are without radioactivity. Requirements for acetylation in vitro of protein in rat liver are different from those in frog liver; GSH has no effect in the rat liver system and is inhibitory in the frog liver system. Among various acetylated proteins, proteins insoluble in 0.1m-sulphuric acid have the highest radioactivity.     

Cations as Mediators of the Adsorption of Nucleic Acids on Clay Surfaces in Prebiotic Environments

Monovalent ([Na⁺] > 10 mM) and divalent ([Ca²⁺], [Mg²⁺] > 1.0 mM) cations induced the precipitationof nucleic acid molecules. In the presence of clay minerals (montmorillonite and kaolinite), there was adsorption instead of precipitation. The cation concentration needed for adsorption depended on both the valence of the cations and the chemical nature of the nucleic acid molecules. Double-stranded nucleic acids needed higher cation concentrations than single-stranded ones to be adsorbed to the same extent on clay. Divalent cations were more efficient than monovalent ones in mediating adsorption. Adsorption to the clay occurred only when both nucleic acids and cations were present. However, once the complexes were formed, the cations could not be removed from the system by washing, indicating that they are directly involved in the association between nucleic acids and mineral surfaces.These observations indicate that cations take part directly in the formation of nucleic acid-clay complexes, acting as a `bridge' between the negative charges on the mineral surface and those of the phosphate groups of the genetic polymer. The relatively low cation concentrations needed for adsorption and the ubiquitous presence of clay minerals in the environment suggest that the adsorption of nucleic acids on mineral surfaces could have taken place in prebiotic habitats. This may have played an important role in the formation and preservation of nucleic acids and/or their precursor polymers.