Polypeptide Sequence Research Articles

Virtual Screeningfor Novel HIV-Reverse Transcriptase Inhibitors

HIV–1 (human immunodeficiency virus type–1) isthe disease causing agent for AIDS. It is pathogenic retrovirus which joined in a long polypeptide chain when viral RNA is translated into a polypeptide sequence. It contains different proteins like reverse transcriptase, protease, integrase, etc. These proteins have to be altered from polypeptide chain before these enzymes become to start function. Reverse transcriptase is inhibited by. We applied existing system by virtual screening analysis of HIV-RT from PDB database versus chemical compounds from ZINC database using “Autodockvina” and “cornia sketch tool”. Different hypothetical ligands were deliberate on cornia sketch tool and their structures were docked with (HIV-RT) PDB file. The structure with best recording was selected and the database of zinc was separated for similar structures and consequences in 18 hits.

A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties

This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine.

Exhaustive search of linear information encoding protein-peptide recognition.

High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.

Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells.

To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs). A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Conformation-Specific Infrared and Ultraviolet Spectroscopy of Cold [YAPAA+H]+ and [YGPAA+H]+ Ions: A Stereochemical "Twist" on the β-Hairpin Turn.

Incorporation of the unnatural d-proline (DP) stereoisomer into a polypeptide sequence is a typical strategy to encourage formation of β-hairpin loops because natural sequences are often unstructured in solution. Using conformation-specific IR and UV spectroscopy of cold (≈10 K) gas-phase ions, we probe the inherent conformational preferences of the DP and LP diastereomers in the protonated peptide [YAPAA+H]+, where only intramolecular interactions are possible. Consistent with the solution-phase studies, one of the conformers of [YADPAA+H]+ is folded into a charge-stabilized β-hairpin turn. However, a second predominant conformer family containing two sequential γ-turns is also identified, with similar energetic stability. A single conformational isomer of the LP diastereomer, [YALPAA+H]+, is found and assigned to a structure that is not the anticipated "mirror image" β-turn. Instead, the LP stereocenter promotes a cis-alanine-proline amide bond. The assigned structures contain clues that the preference of the DP diastereomer to support a trans-amide bond and the proclivity of LP for a cis-amide bond is sterically driven and can be reversed by substituting glycine for alanine in position 2, forming [YGLPAA+H]+. These results provide a basis for understanding the residue-specific and stereospecific alterations in the potential energy surface that underlie these changing preferences, providing insights to the origin of β-hairpin formation.

Hyriopsis cumingii Hic52—A novel nacreous layer matrix protein with a collagen-like structure

Nacre is a product of a precisely regulated biomineralization process and a major contributor to the luster of pearls. Nacre is composed of calcium carbonate and an organic matrix of proteins that is secreted from mollusc mantle tissue and is exclusively associated with shell formation. In this study, hic52, a novel matrix protein gene from mantle of Hyriopsis cumingii, was cloned and functionally analyzed. The full-length cDNA of hic52 encoded 542 amino acids and contained a signal peptide of 18 amino acids. Excluding the signal peptide, the theoretical molecular mass of the polypeptide was 52.2kDa. The predicted isoelectric point was 10.37, indicating a basic shell protein. The amino acid sequence of hic52 featured high proportion of Gly (28.8%) and Gln (12.4%) residues. The predicted tertiary structure was characterized as having similarities to collagen I, alpha 1 and alpha 2 in the structure. The polypeptide sequence shared no homology with collagen. The hic52 expression pattern by quantitative real-time PCR and in situ hybridization exhibits at the dorsal epithelial cells of the mantle. Expression increased during the stages of pearl sac development. The data showed that hic52 is probably a framework shell protein that mediates and controls the nacreous biomineralization process.

CDNA cloning and characterization of UDP‐glucosyltransferase from Indigofera tinctoria

Indigofera tinctoria is a cultivated plant that is utilized as a source of the natural blue dye, indigo. The plant produces a colorless secondary metabolite known as indican (indoxyl β‐D‐glucoside). Following its released from the cells, indican is enzymatically degraded into indoxyl and glucose. Indoxyl is an unstable compound that is easily oxidized and dimerizes to produce the stable indigo. Unlike the degradation reaction of indican, there is limited knowledge about indican biosynthesis pathway in the cells. We have previously reported that Polygonum tinctorium, an alternative indigo‐producing plant, has UDP‐glucosyltransferase (UGT) that catalyzes the synthesis reaction of indican from indoxyl and UDP‐glucose. However, there are several subjects that need to be studied including the cDNA cloning, the subcellular localization, the transport pathway of substrate and product during indican metabolism, etc. We hypothesize that the indican metabolic pathway in Indigofera is similar to that in Polygonum. Here, we report cDNA cloning and characterization of UGT from Indigofera tinctoria. The sample containing UGT that was partially purified from Indigofera leaves was separated on SDS‐PAGE gel. After staining the gel with CBB, several bands were cut off and analyzed by peptide mass fingerprinting. We identified two fragments homologous to UGT from the transcriptomic data that was generated from Indigofera leaf tissue. Based on the sequence of the fragments, RACE method was performed, leading to amplification of two types of full‐length Ugt cDNAs, itUgt1 and itUgt2. The polypeptide sequence of 477 amino acids encoded by itUgt1 shows high homology (max identity is 89%) with the 475 amino acids encoded by itUgt2. Their primary structures contain the motif of UDP‐glucoronosyl and ‐glucosyl transferase and show the high homology to some UGTs from other plants. To analyze the properties and functions of itUGT1 and itUGT2, the recombinant proteins were expressed in E. coli and purified. The antibody was raised against recombinant itUGT2. The recombinant itUGT1 and itUGT2 show high activity of indican synthesis as reported for the Polygonum enzyme. Although there was no difference between values of optimum pH, itUGT2 was more stable with temperature variations than itUGT1. Further, the expression of itUGT proteins and mRNA levels in various tissues from Indigofera were examined by Western blot and semi‐quantitative RT‐PCR, respectively. Consequently, it was observed that UGT1 synthesizes indican in Indigofera tissues and not UGT2. Further work on cell fractionation, for determining the subcellular localization of itUGTs, is in progress

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration.

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency.

Improving the Performance of Simulations of the Intrinsically Disordered N-Terminal Domain from P53

Signal transduction in intrinsically disordered proteins (IDPs) is frequently understood in the context of binding-induced folding, but accurate simulations of IDP structures remain computationally challenging. Protein-solvent interactions, electrostatics, and prolyl isomerization all contribute to the complexity of the behavior of IDPs in solution. Previous studies have indicated a relationship between local alterations in Polyproline-II (PPII) propensities and hydrodynamic radius (RH) which serves as an indicator of global structural changes. Unfortunately, extension of these studies to long polypeptide sequences is computationally prohibitive. We are interested in the behavior of the intrinsically disordered N-terminal domain (NTD) of the p53 protein, a 93-residue region involved in multiple protein binding and cell cycle control. In previous work, accurate simulation of hydrodynamic properties has been limited to simple sequences of 50 residues or less, excluding the possibility of modeling the full length NTD. This study builds upon established computational methods to create a more efficient Monte-Carlo simulation of the NTD that retains the chemically-realistic solvation energy calculations employed previously. Our modified simulation is shown to reproduce faithfully the hydrodynamic properties of the NTD, while at the same time running significantly faster for polypeptides larger than 80 amino acids.

Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin

Microtubules are part of a dynamic cytoskeletal network that is constantly being reorganized to control cell processes such as neuronal development and maintenance, cell division, and cargo transport. Many stabilizing and destabilizing enzymes function to reorganize these networks for the specific needs of the cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules. Our lab has previously shown free tubulin dimers and CTTs alone can inhibit katanin severing. We seek to determine the manner that tubulin CTTs sequence can regulate katanin activity using polypeptide sequences of CTTs of different tubulin isoforms. We find that the sequence's ionic, hydrophobic, and steric features play a role in determining katanin's activity.

The Complete Genome Sequence of a Second Distinct Betabaculovirus from the True Armyworm, Mythimna unipuncta.

The betabaculovirus originally called Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the characteristic size range and morphology of betabaculovirus granules. Barcoding PCR using cytochrome oxidase I primers with DNA from the MyspGV#8 collection sample confirmed that it had been isolated from the true armyworm, Mythimna unipuncta (Lepidoptera: Noctuidae) and therefore was renamed MyunGV#8. The MyunGV#8 genome was found to be 144,673 bp in size with a nucleotide distribution of 49.9% G+C, which was significantly smaller and more GC-rich than the genome of Pseudaletia unipuncta granulovirus H (PsunGV-H), another M. unipuncta betabaculovirus. A phylogeny based on concatenated baculovirus core gene amino acid sequence alignments placed MyunGV#8 in clade a of genus Betabaculovirus. Kimura-2-parameter nucleotide distances suggested that MyunGV#8 represents a virus species different and distinct from other species of Betabaculovirus. Among the 153 ORFs annotated in the MyunGV#8 genome, four ORFs appeared to have been obtained from or donated to the alphabaculovirus lineage represented by Leucania separata nucleopolyhedrovirus AH1 (LeseNPV-AH1) during co-infection of Mythimna sp. larvae. A set of 33 ORFs was identified that appears only in other clade a betabaculovirus isolates. This clade a-specific set includes an ORF that encodes a polypeptide sequence containing a CIDE_N domain, which is found in caspase-activated DNAse/DNA fragmentation factor (CAD/DFF) proteins. CAD/DFF proteins are involved in digesting DNA during apoptosis.

Proteomic analysis of isozymes in Butea superba leaves

The proliferation of protein abundances under influence of seasonal changes are involved in cellular plant metabolic pathways. The protein profiles in relevant with seasonal variations in Butea superba leaves collected in winter, summer and rainy season were evaluated by two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupoletime- of-flight tandem mass spectrometry. The 84 proliferated protein spots were sum up of total proteins, which were found in 3 season-collected plant leaves and were classified into 11 functional groups. Eight proteins which exhibited the alteration of abundant levels in different seasons were found involved in carbohydrate and energy metabolism, photosynthesis, secondary metabolism, stress, RNA metabolism, ROS scavenging and detoxifying, and protein destination and storage. There were 2 proteins exhibited obviously isozyme polypeptide sequences. The variable physiological status within the plant leaf might be influenced by the quantitative and qualitative proliferated protein presences with response to seasonal changes and thus allowing plant to survive during severe abiotic stresses during seasonal changes.

Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences

Key messageRice prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of prolamins that must accumulate in PB-Is.Rice seed prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice prolamins, we compared 13a and 13b prolamins because the amino acid sequences of these prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.

PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress.

Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.

Analysis of Shannon entropy for protein crystallization and prediction of crystallization pH and precipitants

This is a new attempt at analysing crystallization data from Protein Data Bank. In line with the idea that crystallization conditions are intimately related with amino acid sequences, we have computed the Shannon entropy of polypeptides and polynucleotides and we have discovered a relationship between Shannon entropy and molecular weight, and also between the entropy of polypeptides, and the precipitants used in crystallization experiments. In fact, the Shannon entropy and the molecular weight of proteins are good precipitant predictors. On the other hand, we have proposed the hypothesis that homologous proteins may have similar crystallization conditions, and we have tried to find evidence that supports it, predicting the crystallization pH of a large amount of polypeptide sequences by means of a nearest neighbour approach combined with local sequence alignment.

Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with pulmonary hypertension

The availability of whole genome sequence (WGS) data has made it possible to discover protein variantsin silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in global beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene (EPAS1), a gene associated with pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded byEPAS1(E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The sixEPAS1missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact ofEPAS1alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitatesin silicoidentification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with pulmonary hypertension

The availability of whole genome sequence (WGS) data has made it possible to discover protein variants in silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in global beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene ( EPAS1), a gene associated with pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded by EPAS1 (E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The six EPAS1 missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact of EPAS1 alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitates in silico identification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library.

A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of Km and Vmax values toward agarose were 4.6 mg·mL−1 and 967.5 μM·min−1·mg−1, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries.

Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with high-altitude pulmonary hypertension.

The availability of whole genome sequence (WGS) data has made it possible to discover protein variants in silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in U.S. beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene ( EPAS1), a gene associated with high-altitude pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded by EPAS1 (E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The six EPAS1 missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact of EPAS1 alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitates in silico identification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

Resistance to Macrolide Antibiotics in Public Health Pathogens.

Macrolide resistance mechanisms can be target-based with a change in a 23S ribosomal RNA (rRNA) residue or a mutation in ribosomal protein L4 or L22 affecting the ribosome's interaction with the antibiotic. Alternatively, mono- or dimethylation of A2058 in domain V of the 23S rRNA by an acquired rRNA methyltransferase, the product of an erm (erythromycin ribosome methylation) gene, can interfere with antibiotic binding. Acquired genes encoding efflux pumps, most predominantly mef(A) + msr(D) in pneumococci/streptococci and msr(A/B) in staphylococci, also mediate resistance. Drug-inactivating mechanisms include phosphorylation of the 2'-hydroxyl of the amino sugar found at position C5 by phosphotransferases and hydrolysis of the macrocyclic lactone by esterases. These acquired genes are regulated by either translation or transcription attenuation, largely because cells are less fit when these genes, especially the rRNA methyltransferases, are highly induced or constitutively expressed. The induction of gene expression is cleverly tied to the mechanism of action of macrolides, relying on antibiotic-bound ribosomes stalled at specific sequences of nascent polypeptides to promote transcription or translation of downstream sequences.