STAR: predicting recombination sites from amino acid sequence

Background: Designing novel proteins with site-directed recombination has enormous prospects. By locating effective recombination sites for swapping sequence parts, the probability that hybrid sequences have the desired properties is increased dramatically. The prohibitive requirements for applying current tools led us to investigate machine learning to assist in finding useful recombination sites from amino acid sequence alone. Results: We present STAR, Site Targeted Amino acid Recombination predictor, which produces a score indicating the structural disruption caused by recombination, for each position in an amino acid sequence. Example predictions contrasted with those of alternative tools, illustrate STAR'S utility to assist in determining useful recombination sites. Overall, the correlation coefficient between the output of the experimentally validated protein design algorithm SCHEMA and the prediction of STAR is very high (0.89). Conclusion: STAR allows the user to explore useful recombination sites in amino acid sequences with unknown structure and unknown evolutionary origin. The predictor service is available from http://pprowler.itee.uq.edu.au/star.

The effect of box shape on the dynamic properties of proteins simulated under periodic boundary conditions

The effect of the box shape on the dynamic behavior of proteins simulated under periodic boundary conditions is evaluated. In particular, the influence of simulation boxes defined by the near-densest lattice packing (NDLP) in conjunction with rotational constraints is compared to that of standard box types without these constraints. Three different proteins of varying size, shape, and secondary structure content were examined in the study. The statistical significance of differences in RMSD, radius of gyration, solvent-accessible surface, number of hydrogen bonds, and secondary structure content between proteins, box types, and the application or not of rotational constraints has been assessed. Furthermore, the differences in the collective modes for each protein between different boxes and the application or not of rotational constraints have been examined. In total 105 simulations were performed, and the results compared using a three-way multivariate analysis of variance (MANOVA) for properties derived from the trajectories and a three-way univariate analysis of variance (ANOVA) for collective modes. It is shown that application of roto-translational constraints does not have a statistically significant effect on the results obtained from the different simulations. However, the choice of simulation box was found to have a small (5-10%), but statistically significant effect on the behavior of two of the three proteins included in the study. (c) 2005 Wiley Periodicals, Inc.

Predicting structural disruption of proteins caused by crossover

We present a machine learning model that predicts a structural disruption score from a protein’s primary structure. SCHEMA was introduced by Frances Arnold and colleagues as a method for determining putative recombination sites of a protein on the basis of the full (PDB) description of its structure. The present method provides an alternative to SCHEMA that is able to determine the same score from sequence data only. Circumventing the need for resolving the full structure enables the exploration of yet unresolved and even hypothetical sequences for protein design efforts. Deriving the SCHEMA score from a primary structure is achieved using a two step approach: first predicting a secondary structure from the sequence and then predicting the SCHEMA score from the predicted secondary structure. The correlation coefficient for the prediction is 0.88 and indicates the feasibility of replacing SCHEMA with little loss of precision. ©2005 IEEE

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Responsive hydrophobically associating polymers:a review of structure and properties

Responsive hydrophobically associating polymers can in many ways be considered to be analogous to proteins in their ability to form compact molecules with a defined secondary structure, and hence, functionality. These molecules are characterized by the presence of alternating charged and hydrophobic groups. The balance between charge repulsion and hydrophobic interactions is sensitive to environmental pH and therefore changes in pH produce controllable conformational changes. The change from a charged extended chain to a collapsed uncharged coil structure is sometimes referred to as hypercoiling behaviour and enables the polymer to act as a simple switch between an 'on' and 'off' state. The purpose of this review is to illustrate the structure and behaviour of polymers that exhibit hypercoiling behaviour and to highlight their potential pharmaceutical applications, which in terms of drug delivery is likely to be related to their surface behaviour and solubilizing activity. © 2001 Elsevier Science B.V. All rights reserved.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Hybrid Diphenylalkyne-Dipeptide Oligomers Induce Multistrand β-Sheet Formation

Functionalized diphenylalkynes provide a template for the presentation of protein-like surfaces composed of multistrand β-sheets. The conformational properties of three-, four-, and seven-stranded systems have been investigated in the solid- and solution-state. This class of molecule may be suitable for the mediation of therapeutically relevant protein-protein interactions.

Rola białka Hfq w oddziaływaniu niekodujących RNA bakterii z sekwencją kodującą mRNA

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Coarse Graining to Invesitigate Peptide-Lipid Interactions

Experimental characterization of molecular details is challenging, and although single molecule experiments have gained prominence, oligomer characterization remains largely unexplored. The ability to monitor the time evolution of individual molecules while they self assemble is essential in providing mechanistic insights about biological events. Molecular dynamics (MD) simulations can fill the gap in knowledge between single molecule experiments and ensemble studies like NMR, and are increasingly used to gain a better understanding of microscopic properties. Coarse-grained (CG) models aid in both exploring longer length and time scale molecular phenomena, and narrowing down the key interactions responsible for significant system characteristics. Over the past decade, CG techniques have made a significant impact in understanding physicochemical processes. However, the realm of peptide-lipid interfacial interactions, primarily binding, partitioning and folding of amphipathic peptides, remains largely unexplored compared to peptide folding in solution. The main drawback of existing CG models is the inability to capture environmentally sensitive changes in dipolar interactions, which are indigenous to protein folding, and lipid dynamics. We have used the Drude oscillator approach to incorporate structural polarization and dipolar interactions in CG beads to develop a minimalistic peptide model, WEPPROM (Water Explicit Polarizable PROtein Model), and a lipid model WEPMEM (Water Explicit Polarizable MEmbrane Model). The addition of backbone dipolar interactions in a CG model for peptides enabled us to achieve alpha-beta secondary structure content de novo, without any added bias. As a prelude to studying amphipathic peptide-lipid membrane interactions, the balance between hydrophobicity and backbone dipolar interactions in driving ordered peptide aggregation in water and at a hydrophobic-hydrophilic interface, was explored. We found that backbone dipole interactions play a crucial role in driving ordered peptide aggregation, both in water and at hydrophobic-hydrophilic interfaces; while hydrophobicity is more relevant for aggregation in water. A zwitterionic (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and an anionic lipid (POPS: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) are used as model lipids for WEPMEM. The addition of head group dipolar interactions in lipids significantly improved structural, dynamic and dielectric properties of the model bilayer. Using WEPMEM and WEPPROM, we studied membrane-induced peptide folding of a cationic antimicrobial peptide with anticancer activity, SVS-1. We found that membrane-induced peptide folding is driven by both (a) cooperativity in peptide self interaction and (b) cooperativity in membrane-peptide interactions. The dipolar interactions between the peptide and the lipid head-groups contribute to stabilizing folded conformations. The role of monovalent ion size and peptide concentration in driving lipid domain formation in anionic/zwitterionic lipid mixtures was also investigated. Our study suggest monovalent ion size to be a crucial determinant of interaction with lipid head groups, and hence domain formation in lipid mixtures. This study reinforces the role of dipole interactions in protein folding, lipid membrane properties, membrane induced peptide folding and lipid domain formation. Therefore, the models developed in this thesis can be used to explore a multitude of biomolecular processes, both at longer time-scales and larger system sizes.

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. (C) 2010 Elsevier B.V. All rights reserved.

Chiroptical spectroscopy of biomolecules using chiral plasmonic nanostructures

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.

Estudio de las modificaciones conformacionales y su influencia en la actividad inmunológica de péptidos derivados de las familias de las proteínas spect-1 y -2, siap-1 y -2 de plasmodium falciparum

SPECT-1 y -2 y SIAP-1 y -2 son proteínas pertenecientes al esporozoíto de Plasmodium falciparum causante de la malaria más agresiva en los humanos. Estas proteínas están involucradas en el paso del parásito a través de las células del hospedero humano y en la invasión del hepatocito, haciéndolas blancos atractivos para ser estudiadas. Péptidos conservados de alta capacidad de unión (cHABPs) a células HeLa y HepG2 derivados de estas moléculas son no inmunogénicos porque son incapaces de generar una respuesta inmunitaria, pero son claves para el parásito porque cumplen una función importante durante la infección del hospedero humano. En este trabajo se encontró que algunos cHABPs pertenecientes a las proteínas SPECT-1 y -2, están posiblemente involucrados con la unión y formación de poros sobre la membrana de las células hospederas, ayudando al esporozoíto a abrirse paso través de las células del hospedero. Por otro lado, con el fin de cambiar el comportamiento inmunológico de cHABPs derivados de SPECT-1 y -2 y SIAP-1 y -2, se obtuvieron nuevos péptidos mediante el reemplazo de aminoácidos críticos por otros residuos cuya masa molecular sea similar, pero diferente en su polaridad. En este trabajo se reporta que dichas modificaciones promovieron cambios en la estructura secundaria (determinada por DC o 1H-RMN) de los péptidos modificados (mHABPs) cuando se comparó con la estructura de los cHABPs nativos; adicionalmente, estos mHABPs invirtieron su comportamiento inmunológico convirtiéndose en péptidos inmunogénicos inductores de anticuerpos. Lo que permite establecer la existencia de una relación entre la estructura que adoptan estos mHABPs con su actividad inmunológica. Además, algunos de los mHABPs estudiados aquí, pueden ser candidatos a ser incluidos en la vacuna contra la malaria químicamente sintetizada multi-epitope y multi-estadio que se está desarrollando en la Fundación Instituto de Inmunología de Colombia (FIDIC).

miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data

miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This article describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/research/software/mirdeep-star

miRPlant : an integrated tool for identification of plant miRNA from RNA sequencing data

Background Small RNA sequencing is commonly used to identify novel miRNAs and to determine their expression levels in plants. There are several miRNA identification tools for animals such as miRDeep, miRDeep2 and miRDeep*. miRDeep-P was developed to identify plant miRNA using miRDeep’s probabilistic model of miRNA biogenesis, but it depends on several third party tools and lacks a user-friendly interface. The objective of our miRPlant program is to predict novel plant miRNA, while providing a user-friendly interface with improved accuracy of prediction. Result We have developed a user-friendly plant miRNA prediction tool called miRPlant. We show using 16 plant miRNA datasets from four different plant species that miRPlant has at least a 10% improvement in accuracy compared to miRDeep-P, which is the most popular plant miRNA prediction tool. Furthermore, miRPlant uses a Graphical User Interface for data input and output, and identified miRNA are shown with all RNAseq reads in a hairpin diagram. Conclusions We have developed miRPlant which extends miRDeep* to various plant species by adopting suitable strategies to identify hairpin excision regions and hairpin structure filtering for plants. miRPlant does not require any third party tools such as mapping or RNA secondary structure prediction tools. miRPlant is also the first plant miRNA prediction tool that dynamically plots miRNA hairpin structure with small reads for identified novel miRNAs. This feature will enable biologists to visualize novel pre-miRNA structure and the location of small RNA reads relative to the hairpin. Moreover, miRPlant can be easily used by biologists with limited bioinformatics skills.

A comparative analysis of Mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles

Coleoptera is the most diverse group of insects with over 360,000 described species divided into four suborders: Adephaga, Archostemata, Myxophaga, and Polyphaga. In this study, we present six new complete mitochondrial genome (mtgenome) descriptions, including a representative of each suborder, and analyze the evolution of mtgenomes from a comparative framework using all available coleopteran mtgenomes. We propose a modification of atypical cox1 start codons based on sequence alignment to better reflect the conservation observed across species as well as findings of TTG start codons in other genes. We also analyze tRNA-Ser(AGN) anticodons, usually GCU in arthropods, and report a conserved UCU anticodon as a possible synapomorphy across Polyphaga. We further analyze the secondary structure of tRNA-Ser(AGN) and present a consensus structure and an updated covariance model that allows tRNAscan-SE (via the COVE software package) to locate and fold these atypical tRNAs with much greater consistency. We also report secondary structure predictions for both rRNA genes based on conserved stems. All six species of beetle have the same gene order as the ancestral insect. We report noncoding DNA regions, including a small gap region of about 20 bp between tRNA-Ser(UCN) and nad1 that is present in all six genomes, and present results of a base composition analysis.