Primary Structure Of Protein Research Articles

Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization

Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into “attachment discs” that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (∼100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin type, while motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive “linker”, which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers.

New Genetic Variation in BCR gene of Major B3a2 Breakpoint BCR-ABL Fusion Gene in Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia

Background:Polymorphic bases in several exons of the BCR gene have been found in several studies of the BCR-ABL fusion gene. Most of the polymorphisms do not have any implications for the primary structure of the BCR-ABL protein. Nucleotide changes are often located in the area close to the fusion region, and therefore may influence primer annealing. Our previous work failed to amplify 15 of 200 samples from BCR-ABL positive chronic myelogenous leukemia (CML) patients using multiplex PCR, the standard method to detect BCR-ABL transcripts used in our institution. The failure was considered due to problems in primer annealing caused by sequence variations. Sequence analysis of BCR-ABL fusion gene breakpoint types in CML patients has never been hitherto performed in Indonesia. Therefore, the aim of this study was to perform sequence analysis of several samples that did not show amplification using the standard method.Methods:Fifteen samples were qualitatively amplified by two-step PCR using inner primers in the 2nd PCR to determine the breakpoint type of the BCR-ABL fusion gene. The 2nd PCR products were used as templates to perform sequence analysis, and the results were compared to those in genbank.Result:Seven and 5 of 15 samples were confirmed as major b3a2 and major b2a2, respectively. One sample featured a combination of b3a2 and b2a2, and 2 samples a combination of b3a2 and b2a2 with an additional fragment at 500bp. Sequence analysis showed 3 sequence variations in the major b3a2 breakpoint. One had been reported earlier (c.3296T>C) but the others (c.3245C>T and c.3359T>C) were novel. Fragments at 500bp were confirmed as b3a2 and similar sequence b3a2 in genbank.Conclusion:This study found two new genetic variations in the BCR gene in BCR-ABL fusion cases.

Impact of modifying the primary structure of cytoplasmic and membrane proteins of the placenta on the development of its insufficiency

Impact of modifying the primary structure of cytoplasmic and membrane proteins of the placenta on the development of its insufficiency

T-Cell Proliferation Assay: Determination of Immunodominant T-Cell Epitopes of Food Allergens.

Characterization of allergen-specific T cells is critical to understand their contribution to disease pathogenesis. The identification of immunodominant T-cell epitopes is crucial for development of T-cell-based vaccines. Peptide-specific T-cell proliferation studies are usually performed in a library of short synthetic peptides (15mer or 20mer) with 3 or 5 offset spanning the entire length of the allergen. T-cell peptide epitopes lack the primary and tertiary structure of the native protein to cross-link IgE, but retain the ability to stimulate T cells. The peptides sequences can also be obtained either by in silico approaches and in vitro binding assays. The efficacy of T-cell epitope-based peptide immunotherapy has been proven in certain allergies. The present methodology describes T-cell proliferation assays using whole blood sample from allergic subjects.

HOMOLOGY BETWEEN SEGMENTS OF HUMAN HEMOSTATIC PROTEINS AND PROTEINS OF VIRUSES WHICH CAUSE ACUTE RESPIRATORY INFECTIONS OR DISEASES WITH SIMILAR SYMPTOMS

Objectives: To identify homologous segments of human hemostatic and viral proteins and to assess the role of human hemostatic proteins in viral replication. Materials and Methods: The following viruses were chosen for comparison: influenza B (B/Astrakhan/2/2017), coronaviruses (Hcov229E and SARS-Co), type 1 adenovirus (adenoid 71), measles (ICHINOSE-BA) and rubella (Therien). The primary structures of viral proteins and 41 human hemostatic proteins were obtained from open–access www.ncbi.nlm.nih. gov and www.nextprot.org databases, respectively. Sequence homology was determined by comparing 12-amino-acid segments. Those sequences identical in ≥ 8 positions were considered homologous. Results: The analysis shows that viral proteins contain segments which mimic a number of human hemostatic proteins. Most of these segments, except those of adenovirus proteins, are homologous with coagulation factors. The increase in viral virulence, as in case of SARS-Co, correlates with an increased number of segments homologous with hemostatic proteins. Conclusion: Hemostasis plays an important role in viral replication and pathogenesis. The conclusion is consistent with the literature data about the relationship of hemostasis and inflammatory response to viral infections.

Prediction of protein thermostability from their primary structure: the current state and development factors

The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.

Identification and characterization of a gamma-interferon-inducible lysosomal thiol reductase homolog from guinea pig (Cavia porcellus) that exhibits thiol reductase activity in vitro

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is a key enzyme in the antigen processing and presentation pathway whereby it reduces disulfide bonds at an acidic pH. In this study, a homolog of GILT from guinea pigs (designated gpGILT) was identified and characterized using bioinformatic methods and bioactivity assays. The open reading frame of gpGILT is 705bp in length and encodes 234 amino acids, with a putative molecular weight of about 25.85kDa. The structure of gpGILT is similar to those of humans and zebrafish, containing six introns and seven exons. The deduced primary structure of the gpGILT protein includes all of the typical features of other known GILT proteins, including an active-site motif, CXXC, a GILT signature sequence, CQHGX2ECX2NX4C, three potential Asn-linked glycosylation sites, and six other conserved cysteines. The predicted tertiary structures of gpGILT, human GILT, and mouse GILT are quite similar in shape and positional arrangement of the key motifs modeled on the same template. Amino acid sequence-based alignment and phylogenetic analysis showed that gpGILT is most closely related to that from the rat, with an identity of 68.40%. Additionally, the constitutive expression and immune response to lipopolysaccharide (LPS) challenge of gpGILT were tested using real-time quantitative polymerase chain reaction. A tissue-specific expression pattern in selected tissues and remarkable up-regulation of gpGILT mRNA in spleen and blood within 12h of LPS stimulation were observed, suggesting that GILT functions as an immunological surveillance-related factor in both innate and adaptive immunity. Soluble recombinant gpGILT produced in E. coli could reduce the interchain disulfide bonds of IgG in an acidic reaction system in vitro, suggesting thiol reductase activity in antigen processing. The results of this study provide a better understanding of the molecular characteristics of gpGILT and are a useful reference for further investigation of its involvement in antigen processing and immunological surveillance using the laboratory guinea pig.

Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein.

Despite the growing application of gas-phase measurements in structural biology and drug discovery, the factors that govern protein stabilities and structures in a solvent-free environment are still poorly understood. Here, we examine the solvent-free unfolding pathway for a group of homologous serum albumins. Utilizing a combination of chemical probes and noncovalent reconstructions, we draw new specific conclusions regarding the unfolding of albumins in the gas phase, as well as more general inferences regarding the sensitivity of collision induced unfolding to changes in protein primary and tertiary structure. Our findings suggest that the general unfolding pathway of low charge state albumin ions is largely unaffected by changes in primary structure; however, the stabilities of intermediates along these pathways vary widely as sequences diverge. Additionally, we find that human albumin follows a domain associated unfolding pathway, and we are able to assign each unfolded form observed in our gas-phase data set to the disruption of specific domains within the protein. The totality of our data informs the first detailed mechanism for multidomain protein unfolding in the gas phase, and highlights key similarities and differences from the known solution-phase pathway.

Utilization of Hydrogen/Deuterium Exchange in Biopharmaceutical Industry

The development of biopharmaceutics is the fastest growing segment of the present pharmaceutical industry. The analysis of proteins therapeutics is a challenging task due to their large size and complexity of spatial structure. Any changes in the primary, secondary, tertiary or quaternary protein structure can have huge impact on their function, efficiency and toxicity. Mass spectrometry proved itself to be a powerful tool for analysis of primary protein structure (amino acid sequence) and thanks to the development of new techniques in last years it is able to analyse higher order protein structures. One of these new techniques is hydrogen/deuterium exchange (HDX). HDX is based on exchange of amid protons with deuterium from solution on the protein backbone chain. Protons on the surface of protein are exchanging with deuterium much faster than protons buried inside of protein. HDX results could provide information about spatial protein structure and also about protein-protein interactions and protein-ligand interactions. Furthermore, by analysing of deuterium exchange in different time points this method could give information about dynamic changes of protein structure and dynamics of proteins interactions. Because of possibilities of this method, HDX become attractive method for characterization of protein biopharmaceuticals. This review article is focused on the utilization of mass spectrometry in biopharmaceutical industry and mainly on HDX method and its applications.Key words: mass spectrometry - proteomics - protein conformation - drug discovery - drug industry - hydrogen/deuterium exchangeThe work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 23. 5. 2016Accepted: 10. 6. 2016.

Gene regulation and genetics in neurochemistry, past to future.

Ask any neuroscientist to name the most profound discoveries in the field in the past 60years, and at or near the top of the list will be a phenomenon or technique related to genes and their expression. Indeed, our understanding of genetics and gene regulation has ushered in whole new systems of knowledge and new empirical approaches, many of which could not have even been imagined prior to the molecular biology boon of recent decades. Neurochemistry, in the classic sense, intersects with these concepts in the manifestation of neuropeptides, obviously dependent upon the central dogma (the established rules by which DNA sequence is eventually converted into protein primary structure) not only for their conformation but also for their levels and locales of expression. But, expanding these considerations to non-peptide neurotransmitters illustrates how gene regulatory events impact neurochemistry in a much broader sense, extending beyond the neurochemicals that translate electrical signals into chemical ones in the synapse, to also include every aspect of neural development, structure, function, and pathology. From the beginning, the mutability - yet relative stability - of genes and their expression patterns were recognized as potential substrates for some of the most intriguing phenomena in neurobiology - those instances of plasticity required for learning and memory. Near-heretical speculation was offered in the idea that perhaps the very sequence of the genome was altered to encode memories. A fascinating component of the intervening progress includes evidence that the central dogma is not nearly as rigid and consistent as we once thought. And this mutability extends to the potential to manipulate that code for both experimental and clinical purposes. Astonishing progress has been made in the molecular biology of neurochemistry during the 60years since this journal debuted. Many of the gains in conceptual understanding have been driven by methodological progress, from automated high-throughput sequencing instruments to recombinant-DNA vectors that can convey color-coded genetic modifications in the chromosomes of live adult animals. This review covers the highlights of these advances, both theoretical and technological, along with a brief window into the promising science ahead. This article is part of the60th Anniversary special issue.

Insight towards the identification of cytosolic Ca2+ -binding sites in ryanodine receptors from skeletal and cardiac muscle.

Ca2+ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation-contraction (E-C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca2+ elevations. Although this process is not critical for skeletal E-C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca2+ -binding proteins that harbour highly selective Ca2+ -binding sites to receive and translate the various Ca2+ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF-hand nature. EF-hand proteins share a common helix-loop-helix structural motif with highly conserved residues involved in Ca2+ coordination. The first step in predicting EF-hand positive regions is to compare the primary protein structure with the EF-hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca2+ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca2+ -binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.

Effect of transglutaminase-catalyzed glycosylation on the allergenicity and conformational structure of shrimp (Metapenaeus ensis) tropomyosin.

Tropomyosin (TM), a myofibrillar protein, is a major allergen in shrimp. The aim of this study was to evaluate the effect of transglutaminase (TGase)-catalyzed glycosylation on the potential allergenicity and conformational structure of TM in Metapenaeus ensis. Results showed that glycosylation of TM induced unfolding of the primary protein structure followed by loss of the secondary structure. Cleavage of certain free amino groups was observed during TGase-catalyzed glycosylation. The glycosylation rate correlated with reaction temperature. Western blotting and indirect ELISA with TM-specific polyclonal antibodies from rabbit and sera from patients allergic to shrimp demonstrated that antigenicity and potential allergenicity of TM decreased, which correlated well with the conformational changes in its structure. Considering TGase is widely utilized in the food industry, these results indicate that TGase-catalyzed glycosylation has the potential to serve as a mild method for reducing the allergenicity of shrimp products.

The conformational IgE epitope profile of soya bean allergen Gly m 4

Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Protein Primary Structure of the Vaccinia Virion at Increased Resolution

Poxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.

Polymorphism analysis of prion protein gene in 11 Pakistani goat breeds

ABSTRACTThe association between caprine PrP gene polymorphisms and its susceptibility to scrapie has been investigated in current years. As the ORF of the PrP gene is extremely erratic in different breeds of goats, we studied the PrP gene polymorphisms in 80 goats which belong to 11 Pakistani indigenous goat breeds from all provinces of Pakistan. A total of 6 distinct polymorphic sites (one novel) with amino acid substitutions were identified in the PrP gene which includes 126 (A -> G), 304 (G -> T), 379 (A -> G), 414 (C -> T), 428 (A -> G) and 718 (C -> T). The locus c.428 was found highly polymorphic in all breeds as compare to other loci. On the basis of these PrP variants NJ phylogenetic tree was constructed through MEGA6.1 which showed that all goat breeds along with domestic sheep and Mauflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species while Protein analysis has shown that these polymorphisms can lead to varied primary, secondary and tertiary structure of protein. Based on these polymorphic variants, genetic distance, multidimensional scaling plot and principal component analyses revealed the clear picture regarding greater number of substitutions in cattle PrP regions as compared to the small ruminant species. In particular these findings may pinpoint the fundamental control over the scrapie in Capra hircus on genetic basis.

First step towards the biomolecular characterization of Pompia, an endemic Citrus-like fruit from Sardinia (Italy)

This study is the first molecular and biochemical analysis conducted on Pompia, a plant of unknown origin that is endemic to Sardinia; this plant is thought to belong to the Citrus genus. Here, genes coding for the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), and polyphenol oxidase (PPO, EC 1.14.18.1) were identified. We detected the aforementioned enzymes in fresh leaf tissue and assessed the catalytic activity of each to support the molecular and biochemical data. This was the first molecular study to define the primary structure of proteins with antioxidant activity in Pompia. The study also contributed to the enrichment of gene databases and created the basis for molecular phylogenetic studies, which is important because this plant currently has no taxonomic or phylogenetic classification.

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A.

ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor.

Integrative Methods for Elucidating the Structure & Function of Cellular Machines

The myriad events that occur in living cells (replication, organellar assembly, transport, genome organization, transcription etc.) are to a large extent carried out through dynamic associations and assemblies of macromolecules. I will describe our efforts to develop and integrate sets of tools that are designed to throw light on the evolution, structure and function of these macromolecular machines.At the very earliest stage of analysis, we define the precise ensembles of macromolecular players that make up the assemblies of interest. Mass spectrometry has become an increasingly enabling tool for this task, owing to its sensitivity and its ability to characterize the primary structures of endogenous proteins along with any modifications that may be present. At the next stage of analysis, we apply combined proteomic‐biochemical approaches to gain insights into the architectures of protein complexes, especially those that are too large or too poorly behaved for traditional X‐ray crystallographic diffraction or NMR spectroscopic analyses. Finally, we integrate data from all these sources to generate models that can provide insights into the evolution, structure and function of these remarkable macromolecular machines. I will to discuss current capabilities and limits of this approach as well as prospects for the future.Support or Funding InformationThis work was supported in part by NIH grants GM103314 and GM109824.

Suitability of Sequence-Based Feature Vector for Classification Algorithm Improves Accuracy of Human Protein-Protein Interaction Prediction: A Red Blood Cell Case Study

To classify human protein-protein interaction information and consolidate existing data, supervised learning algorithms are implemented. These algorithms require a feature vector to generate a prediction model and feature vectors could be constructed based on various input data. The suitability of feature vector for classification algorithm results in a more predictive model and predictions with higher accuracies based on low-dimension vectors. To investigate the proper combination of feature sets and the algorithms, three feature vectors including AA Frequency, AA Graphical Parameter, and AA Triplex based on the sole knowledge of primary structure of human red blood cell proteins were constructed and then applied to five different classification methods. The results indicated that support vector machine (SVM) algorithm produced the highest accuracy of 84.65% with AA Graphical Parameter feature set while it reached accuracy of 80.65% with AA Triplex feature set. Random forest (RF) achieved high accuracy of 83.69% with all three feature sets on average. Bayesian classifier of TAN performed better than NB using all three features. Artificial neural network (ANN) classifier demonstrated the lowest average accuracy of 76%; however, the performance was comparable with TAN where AA triplex learning feature was used with the accuracy of 77.90%. These figures demonstrated that selecting an appropriate feature set for a classification task results in a higher accuracy with the advantage of utilizing low-dimension feature vectors constructed from more simple data. Keywords: classification algorithms; Protein-protein interaction prediction; sequence-based feature vectors; machine learning; human protein-protein interaction; accuracy of interaction prediction.

EuGene: maximizing synthetic gene design for heterologous expression.

UNLABELLED Numerous software applications exist to deal with synthetic gene design, granting the field of heterologous expression a significant support. However, their dispersion requires the access to different tools and online services in order to complete one single project. Analyzing codon usage, calculating codon adaptation index (CAI), aligning orthologs and optimizing genes are just a few examples. A software application, EuGene, was developed for the optimization of multiple gene synthetic design algorithms. In a seamless automatic form, EuGene calculates or retrieves genome data on codon usage (relative synonymous codon usage and CAI), codon context (CPS and codon pair bias), GC content, hidden stop codons, repetitions, deleterious sites, protein primary, secondary and tertiary structures, gene orthologs, species housekeeping genes, performs alignments and identifies genes and genomes. The main function of EuGene is analyzing and redesigning gene sequences using multi-objective optimization techniques that maximize the coding features of the resulting sequence. AVAILABILITY EuGene is freely available for non-commercial use, at http://bioinformatics.ua.pt/eugene.