Shotgun Strategy Research Articles

A proteomic analysis of engineered tendon formation under dynamic mechanical loading in vitro

Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for 12 weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical Wnt signal pathway) mediated other biological processes.

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.

Revisiting Iodination Sites in Thyroglobulin with an Organ-oriented Shotgun Strategy

Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.

Generation of a BAC-based physical map of the melon genome.

BackgroundCucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has high intra-specific genetic variation, morphologic diversity and a small genome size (450 Mb), which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. A number of genetic and genomic resources have already been developed, such as several genetic maps and BAC genomic libraries. These tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. However, no physical map of any Cucurbitaceae has yet been developed. A project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. A BAC-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced.ResultsA melon physical map was constructed using a 5.7 × BAC library and a genetic map previously developed in our laboratories. High-information-content fingerprinting (HICF) was carried out on 23,040 BAC clones, digesting with five restriction enzymes and SNaPshot labeling, followed by contig assembly with FPC software. The physical map has 1,355 contigs and 441 singletons, with an estimated physical length of 407 Mb (0.9 × coverage of the genome) and the longest contig being 3.2 Mb. The anchoring of 845 BAC clones to 178 genetic markers (100 RFLPs, 76 SNPs and 2 SSRs) also allowed the genetic positioning of 183 physical map contigs/singletons, representing 55 Mb (12%) of the melon genome, to individual chromosomal loci. The melon FPC database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.ConclusionsHere we report the construction of the first physical map of a Cucurbitaceae species described so far. The physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. The data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in Spain, adopting a whole genome shotgun approach based on 454 sequencing data.

The Genome Sequence of Trypanosoma brucei gambiense, Causative Agent of Chronic Human African Trypanosomiasis

Background Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.Methods and FindingsAn improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.ConclusionsThis study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.

Systematic overrepresentation of DNA termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear DNA molecules

BackgroundAnalysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. We surmised that the problem might lie in the DNA shearing conditions used to create clone libraries for genome sequencing.ResultsA shotgun strategy was used to sequence and assemble circular and linear cosmid DNAs sheared using conditions typical for a genome project. The DNA sheared in circular form assembled into a single sequence contig. However, the linearized cosmid produced an incomplete assembly because the two DNA termini, though greatly overrepresented in the clone library used for sequencing, were separated from neighboring sequences by gaps of ~1.4 and 1.8 kb. These gap sizes were reduced, but not eliminated, by shearing the linear cosmid into smaller fragments. Mapping of shearing breakpoints revealed a paucity of breaks in the subterminal regions of the linearized cosmid and also near chromosome ends of the fungus Neurospora crassa.ConclusionTogether, our data indicate that the ends of linear DNA molecules are recalcitrant to hydrodynamic shearing. We propose that this causes DNA termini to be overrepresented in the resulting fragment population but ultimately prevents their incorporation into sequence assemblies.

Proteomic study of Mycoplasma suis using the gel-based shotgun strategy

Mycoplasma suis (M. suis) is an uncultivable pathogen that colonizes the surface of porcine erythrocytes. In the present study, gel-based LC–MS/MS was performed to analyze the proteomic composition of M. suis. We identified 191 proteins with two or more peptides and additional 217 putative proteins with one peptide hit by cross-species searching the Mollicutes protein databases. Resultant proteins were classified based on their molecular functions. The majority of enzymes involved in central carbon metabolic pathways and nucleotide biosynthesis were confirmed by searching the KEGG database. The present study is the first report addressing the proteome of M. suis. Results indicate that the energy source of M. suis might depend mainly on glycometabolism.

Proteome analysis of cartilage endplate in adolescent idiopathic scoliosis.

Objective To identify protein expression in asymmetrically developed intervertebral discs of adolescent idiopathic scoliosis (AIS) patients and try to find out factors that may originate or strengthen the deformity situation. Methods The apex intervertebral discs were harvested from 6 AIS patients during anterior spinal correction surgery procedure. Proteins in cartilage endplate were extracted and we used shotgun strategy to achieve proteomics investigation, the proteins were classified and their functions were investigated. Results Average protein consistency is 3.02 mg/ml, which was tested through Bradford procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that both species and consistency of the proteins from convex side were higher than that from the concave side. More than 100 proteins were identified from the convex sample, and over 60 proteins were identified from concave side, 58 proteins were found to only express in convex side and 10 in concave side. Conclusion Based on the physiological role, the identified proteins in cartilage endplate from AIS patients were classified preliminarily. Some proteins identified only in convex or concave side were strongly believed to play important roles in contribution to asymmetrically developed apex cartilage endplate and related intervertebral discs.

Sequence analysis of a DNA fragment fromSinorhizobium fredii USDA257 which extends the nitrogen fixation host range ofRhizobium species NGR234 to soybean,Glycine max (L.) Merr cultivar Peking

A fragment of DNA (pBTBX) from the genome ofSinorhizobium fredii USDA257 was sequenced by shotgun strategy to identify the potential genes which enabled theRhizobium species NGR234 to fix nitrogen on soybean,Glycine max (L.) Merr cv. Peking. The total length of the cosmid is 32,824 base pairs with a GC content of 61%. A 29 open reading frames (ORF) were identified representing 71.8% (23,574 bp) of the cosmid. Out of these ORF, 96.5% (22,749 bp) were identical and similar to reported and hypothetical genes and proteins. The remaining 3.5% (825 bp) had no apparent similarity to any genes in the data base. Gene and gene products found on the DNA fragment include those involved in the synthesis of FeMo component of nitrogenase, regulation of nitrogen fixation, transport of amino acids and sugars, chemotaxis and transcriptional regulation.

Analysis of the expression protein profiles of lung squamous carcinoma cell using shot-gun proteomics strategy

The aim of this study is to globally screen and identify the expression protein profiles of lung squamous carcinoma cell (SqCC) using shot-gun proteomics strategy and to further analyze function of individual proteins by bioinformatics, which may likely result in the identification of new biomarkers and provide helpful clues for pathogenesis, early diagnosis, and progression of lung SqCC. The specific tumor cells were isolated and collected from the tissues of six patients with lung SqCC by laser capture microdissection (LCM). Total proteins from the LCM cells were extracted, digested with trypsin. The sequence information of resulting peptides was acquired by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (TMS). The global protein profiles of lung SqCC cell were identified with BioworksTM software in IPI human protein database. Cellular component, molecular function, and biological process of the all proteins were analyzed using gene ontology (GO). About 720,000 tumor cells were satisfactorily collected from tissues of six patients with lung SqCC by LCM and the homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. The high resolution profiles including HPLC, full mass spectrum, and tandem mass spectrum were successfully obtained. Database searching of the resulting bimolecular sequence information identified 1982 proteins in all samples. The bioinformatics of these proteins, including amino acids sequence, fraction of coverage, molecular weight, isoelectric point, etc., were analyzed in detail. Among them, the function of most proteins was recognized by using GO. Five candidate proteins, Prohibitin (PHB), Mitogen-activated protein kinase (MAPK), Heat shock protein27 (HSP27), Annexin A1(ANXA1), and High mobility group protein B1 (HMGB1), might play an important role in SqCC genesis, progression, recurrence, and metastasis according to relative literatures. We have successfully isolated the interesting cells and effectively solved the heterogeneous problem of lung SqCC using LCM. The globally expressional proteins of lung SqCC cell were identified by shot-gun proteomics strategy. The five proteins might be hopefully used as markers of lung SqCC.

The tear proteomics analysis of conjunctivochalasis

To analysis tear proteins by using shotgun strategy, and to study pathogenesis mechanism of conjunctivochalasis by comparing tear proteins between the conjunctivochalasis and normal body. Tears were obtained from 8 conjunctivochalasis cases and 8 normal controls. Fifteen microliters of tears was collected by microcapillary tubes from each eye. Shotgun strategy was used for tear protein analysis. Trypsin digestion in-solution, separation of peptide mixture by reverse-phase high-pressure liquid chromatograph (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS/MS) identification and bioinformation analysis were used in this study. Three hundred and fifty-six proteins were excised in conjunctivochalasis patients and 352 proteins in the normal controls, and among them 119 proteins were the same. The functions of tear proteins were classified with GOA, which found some apoptosis regulation proteins and apoptosis related proteins and inflammatory response related proteins in conjuntivochalasis but not in normal controls group. Defensin was also found in conjuntivochalasis. Shotgun strategy can separate and analysis tear proteins effectively, which provide a new method for tear protein component of conjunctivochalasis. The special component in conjunctivochalasis tear show conjunctivochalasis maybe related to cell apoptosis and inflammatory.

Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategy

The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.

Where Do We Go for Atherothrombotic Disease Genetics?

See related articles, pages 1109–1120. Cardiovascular disease (CVD), including coronary artery disease (CAD) with its fatal clinical outcome myocardial infarction (MI) and stroke, remain the leading cause of deaths in western and nonwestern countries.1,2 In the US, CVD claims more lives than cancer, chronic lower respiratory tract diseases and diabetes mellitus combined, accounting for ≈36% of all deaths in 2004 and incurring an estimated direct and indirect cost of ≈432 billion dollars in 2007.2 Stroke and MI are multifactorial traits with complex pathophysiology. Besides other established factors, family history of disease strongly and independently determines CVD risk.3 There is a nearly 5-fold difference in stroke risk between monocygotic and dizygotic twins,4 and with respect to race, stroke incidence is approximately twice as high in blacks compared to whites.5 Marenberg et al,6 in their study on ≈20 000 Swedish twins, demonstrated that the relative risk to die from MI, when the other twin already died from this disease, was twice as large for monozygotic compared to dizygotic twins. Celera Genomics7 and the International Human Genome Project consortium,8 by whole genome shotgun and clone-based sequencing strategies, respectively, proposed 2 versions of the human genome—the diploid genome offering more genetic information than previously anticipated.9 The availability of almost the entire human genome sequence allows for the immediate access to specific target sequences across the human genome. The number of different technologies to explore genetic sequences on a “genome-wide” scale and high-throughput genotyping methods is steadily increasing. Genome-wide approaches are essentially based on the fact that alleles at nearby loci often show strong linkage disequilibrium (LD). Human recombination events occur ubiquitously in short genomic hotspots (1 to 2 kb) every 100 to 200 kb, preferentially outside genes,10 leading to a breakdown of allelic …

Selected reaction monitoring for quantitative proteomics: a tutorial.

Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC-MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed.

The Implications of Proteolytic Background for Shotgun Proteomics

The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. It was found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed. This unexpected complexity of proteomics samples implies substantial technical challenges, explains some perplexing results in the proteomics literature, and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes.

Analysis of human liver proteome using replicate shotgun strategy

In this study, a liquid-based shotgun strategy was used to comprehensively identify the expression of human liver proteome. Proteins were extracted from human liver tissue and digested in-solution. The tryptic digest mixture was desalted and separated by off-line strong cation exchange (SCX) chromatography with a 60-min elution. The MS/MS spectra were acquired in data-dependent mode after an RP chromatographic separation combined with linear IT MS analysis. To obtain the most comprehensive human liver proteome, each SCX fraction was run six times in RPLC MS/MS manner. Finally, more than 6,000,000 MS/MS spectra were collected. Using a relatively strict filter criteria, 24,311 proteins (48.42% of the predicted human proteome from human International Protein Index (IPI) protein database 3.07) corresponding to 13,150 nonredundant proteins were successfully identified, in which 7001 proteins (53.24%) were identified by two or more peptides, which could be considered as a high-confident dataset. Among the 6149 proteins (46.76%) identified by single peptide, 3812 proteins (61.99%) were detected more than twice in six repeated runs. Comparative analysis between different runs shows that the overlap of identified proteins between any two runs ranged from 25 to 44%. Of the nonredundant proteins identified, 8919 proteins (67.83%) were detected more than twice and 4231 proteins (32.17%) were detected only once in six RPLC MS/MS runs. The Gene Ontology annotation shows that the identified proteins come from various subcellular components. In addition, a large number of low abundant proteins were identified. The dynamic range of the approach reached at least nine orders of magnitude by estimating the concentration of proteins.

Comparison of two‐dimensional gel electrophoresis based and shotgun strategies in the study of plasma membrane proteome

Membrane proteins play important roles in various plasma membrane (PM) activities such as signal transduction and cell recognition. However, a comprehensive proteomic study of membrane proteins remains difficult. Different strategies have been employed to study PM proteome, but little effort has been made to systematically evaluate them. In the present work, liver PM was prepared by subcellular fractionation and an aliquot was washed by sodium carbonate. After evaluation of the PM fraction by electron microscopy and Western blotting, proteins in both original and carbonate washed PM were identified by either 2-DE coupled MALDI-TOF-MS or shotgun strategies. Then protein characteristics such as molecular weight, pI, grand average hydrophobicity, subcellular location, and transmembrane domains were systematically compared. The comparative analysis showed that shotgun strategies were more suitable to identify membrane proteins, while 2-DE-based strategies may serve as a complement. Furthermore, carbonate washing obviously enriched the integral membrane proteins. All the results suggested that the strategy combining carbonate washing and shotgun identification was the optimum strategy to study human liver PM proteome. Using this strategy, 260 high-confidence proteins were identified, wherein 139 were integral membrane proteins which had 1-17 transmembrane domains.

A haplome alignment and reference sequence of the highly polymorphic Ciona savignyi genome

The high degree of polymorphism in the genome of the sea squirt Ciona savignyi complicated the assembly of sequence contigs, but a new alignment method results in a much improved sequence.

The Complete Mitochondrial Genome of the Entomopathogenic Nematode Steinernema carpocapsae: Insights into Nematode Mitochondrial DNA Evolution and Phylogeny

We determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy. The 13,925-bp genome contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins and lacks an ORF encoding ATPase subunit 8. Four initiation codons were inferred, TTT, TTA, ATA, and ATT, most of the genes ended with TAA or TAG, and only two had a T as an incomplete stop codon. All predicted tRNAs showed the nonconventional secondary structure typical of Secernentea. Although we were able to fold the sequences of trnN, trnD, and trnC into more conventional cloverleaf structures after adding adjacent nucleotides, northern blot experiments showed that the nonstandard tRNAs are actually expressed. Phylogenetic and comparative analyses showed that the mitochondrial genome of S. carpocapsae is more closely related to the genomes of A. suum and C. elegans than to that of Strongyloides stercoralis. This finding does not support the phylogeny based on nuclear small subunit ribosomal DNA sequences previously published. This discrepancy may result from differential reproductive strategies and/or differential selective pressure acting on nuclear and mitochondrial genes. The distinctive characteristics observed among mitochondrial genomes of Secernentea may have arisen to counteract the deleterious effects of Muller's ratchet, which is probably enhanced by the reproductive strategies and selective pressures referred to above.