Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. We used inverse PCR of non-B microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures integrated at a common ectopic chromosomal site to show that these non-B DNAs generate highly mutagenized eccDNAs by replication-dependent mechanisms. Mutagenesis occurs within the non-B DNAs and extends several kilobases bidirectionally into flanking and nonallelic DNA. Each non-B DNA exhibits a different pattern of mutagenesis, while sister clones containing the same non-B DNA also display distinct patterns of recombination, microhomology-mediated template switching and base substitutions. Mutations include mismatches, short duplications, long nontemplated insertions, large deletions and template switches to sister chromatids and nonallelic chromosomes. Drug-induced replication stress or the depletion of DNA repair factors Rad51, the COPS2 signalosome subunit or POLη change the pattern of template switching and alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA double strand breaks to account for the generation and circularization of mutagenized eccDNAs and the appearance of genomic homologous recombination deficiency (HRD) scars. These results may help to explain the appearance of tumor eccDNAS and their roles in neoantigen production, oncogenesis and resistance to chemotherapy.

Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. In tumors, highly transcribed eccDNAs have been implicated in oncogenesis, neoantigen production and resistance to chemotherapy. Here we show that unstable microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures generate eccDNAs when integrated at a common ectopic site in human cells. These non-B DNA prone microsatellites form eccDNAs by replication-dependent mechanisms. The microsatellite-based eccDNAs are highly mutagenized and display template switches to sister chromatids and to nonallelic chromosomal sites. High frequency mutagenesis occurs within the eccDNA microsatellites and extends bidirectionally for several kilobases into flanking DNA and nonallelic DNA. Mutations include mismatches, short duplications, longer nontemplated insertions and large deletions. Template switching leads to recurrent deletions and recombination domains within the eccDNAs. Template switching events are microhomology-mediated, but do not occur at all potential sites of complementarity. Each microsatellite exhibits a distinct pattern of recombination, microhomology choice and base substitution signature. Depletion of Rad51, the COPS2 signalosome subunit or POLη alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA breaks for the generation and circularization of mutagenized eccDNAs and genomic homologous recombination deficiency (HRD) scars.

Stable G-quadruplex DNA structures promote replication-dependent genome instability.

G-quadruplex (G4)-prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites. Furthermore, we show that DNA double-strand breaks at the ectopic site are dependent on the nuclease Mus81. Hypermutagenesis during sister chromatid repair extends several kilobases from the G4 site and breaks at the G4 site resulting in microhomology-mediated translocations. To determine whether H3 or G4 structures are responsible for homopurine/homopyrimidine tract instability, we derived constructs and cell lines from the PKD1 repeat, which can only form H3 or G4 structures. Under normal growth conditions, we found that G4 cell lines lost the G4 consensus sequence early during clonal outgrowth, whereas H3 cells showed DNA instability early during outgrowth but only lost reporter gene expression after prolonged growth. Thus, both the H3 and G4 non-B conformation DNAs exhibit genomic instability, but they respond differently to endogenous replication stress. Our results show that the outcomes of replication-dependent double-strand breaks at non-B-DNAs model the instability observed in microhomology-mediated break-induced replication (BIR). Marked variability in the frequency of mutagenesis during BIR suggests possible dynamic heterogeneity in the BIR replisome.

MAGI-1 PDZ2 Domain Blockade Averts Adenovirus Infection via Enhanced Proteolysis of the Apical Coxsackievirus and Adenovirus Receptor.

Adenoviruses (AdVs) are etiological agents of gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdVs use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains, PDZ2 and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease the apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2 were synthesized (TAT-E6 and TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by the activation of the regulated intramembrane proteolysis (RIP) pathway through a disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain, and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has additional translational potential for targeting other viral receptors that have PDZ binding domains, such as the angiotensin-converting enzyme 2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8-interacting protein MAGI-1 and decrease AdV transduction in multiple polarized epithelial models. Peptide-induced CAREx8 degradation is triggered by extracellular domain (ECD) shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of the CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication.

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.

Expansions of CNG microsatellite tracts are responsible for several neurodegenerative diseases, including myotonic dystrophy type 1, Huntington disease, and spinocerebellar ataxia type 8. Here we show that expanded (CNG)n repeats are susceptible not only to expansions and contractions, but are prone to DNA double strand breaks following replication stress. We describe a general strategy for the construction of clonal cell lines containing CNG repeats of various lengths, in which the microsatellites are integrated using the yeast FLP recombinase at a single ectopic recombination acceptor site in the HeLa genome. We illustrate two types of (CTG/CAG) cell lines, one of which contains dual fluorescent marker genes flanking the (CTG/CAG) repeat, and one which does not. We show that long CNG repeats are prone to DNA double strand breaks (DSBs) upon exposure of these cell lines to prolonged replication stress.

Allergen homologs in the Euroglyphus maynei draft genome.

Euroglyphus maynei is a house dust mite commonly found in homes worldwide and is the source of allergens that sensitize and induce allergic reactions in humans. It is the source of species-specific allergens as well as allergens that are cross-reactive with the allergens from house dust mites Dermatophagoides farinae and D. pteronyssinus, and the ectoparasitic scabies mite Sarcoptes scabiei. The genomics, proteomics and molecular biology of E. maynei and its allergens have not been as extensively investigated as those of D. farinae, D. pteronyssinus, and S. scabiei where natural and recombinant allergens from these species have been characterized. Until now, little was known about the genome of E. maynei and it allergens but this information will be important for producing recombinant allergens for diagnostic and therapeutic purposes and for understanding the allergic response mechanism by immune effector cells that mediate the allergic reaction. We sequenced and assembled the 59 Mb E. maynei genome to aid the identification of homologs for known allergenic proteins. The predicted proteome shared orthologs with D. farinae and S. scabiei, and included proteins with homology to more than 30 different groups of allergens. However, the majority of allergen candidates could not be assigned as clear orthologs to known mite allergens. The genomic sequence data, predicted proteome, and allergen homologs identified from E. maynei provide insight into the relationships among astigmatid mites and their allergens, which should allow for the development of improved diagnostics and immunotherapy.

Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory.

BACKGROUND: Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose. OBJECTIVE: The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host's protective response. METHODS: An aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies. RESULTS: We identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum. CONCLUSIONS: Thirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite's ability to modulate its host's innate and adaptive immune responses.

Fatty acid binding profile of the liver X receptor α.

Liver X receptor (LXR)α is a nuclear receptor that responds to oxysterols and cholesterol overload by stimulating cholesterol efflux, transport, conversion to bile acids, and excretion. LXRα binds to and is regulated by synthetic (T-0901317, GW3695) and endogenous (oxysterols) ligands. LXRα activity is also modulated by FAs, but the ligand binding specificity of FA and acyl-CoA derivatives for LXRα remains unknown. We investigated whether LXRα binds FA or FA acyl-CoA with affinities that mimic in vivo concentrations, examined the effect of FA chain length and the degree of unsaturation on binding, and investigated whether FAs regulate LXRα activation. Saturated medium-chain FA (MCFA) displayed binding affinities in the low nanomolar concentration range, while long-chain fatty acyl-CoA did not bind or bound weakly to LXRα. Circular dichroic spectra and computational docking experiments confirmed that MCFA bound to the LXRα ligand binding pocket similar to the known synthetic agonist of LXRα (T0901317), but with limited change to the conformation of the receptor. Transactivation assays showed that MCFA activated LXRα, whereas long-chain FA caused no effect. Our results suggest that LXRα functions as a receptor for saturated FA or acyl-CoA of C10 and C12 in length.

Sarcoptes scabiei: genomics to proteomics to biology.

BACKGROUND: The common scabies mite, Sarcoptes scabiei is a cosmopolitan parasite of humans and other mammals. An annotated genome of Sarcoptes scabiei var. canis has been deposited in the National Center for Biotechnology Information (NCBI) and VectorBase and a proteomic analysis of proteins in extracts of mite bodies and eggs from this strain has been reported. Here we mined the data to identify predicted proteins that are known to be involved in specific biological processes in other animals. RESULTS: We identified predicted proteins that are associated with immunomodulation of the host defense system, and biological processes of the mite including oxygen procurement and aerobic respiration, oxidative metabolism, sensory reception and locating a host, neuronal transmission, stressors (heat shock proteins), molting, movement, nutrient procurement and digestion, and excretion and water balance. We used these data to speculate that certain biological processes may occur in scabies mites. CONCLUSION: This analysis helps understand the biology of Sarcoptes scabiei var. canis and adds to the data already available in NCBI and VectorBase.

A Proteomic Analysis of Sarcoptes scabiei (Acari: Sarcoptidae).

The pruritic skin disease scabies is caused by the burrowing of the itch mite Sarcoptes scabiei (De Geer). It is difficult to diagnose this disease because its symptoms often resemble those of other skin diseases. No reliable blood or molecular diagnostic test is available. The aim of this project was to begin to characterize the scabies proteome to identify scabies mite proteins, including those that may be useful in the development of a diagnostic test or vaccine. Various scabies mite extracts were separated by two-dimensional electrophoresis, and 844 Coomassie Blue-stained protein spots were excised, subjected to trypsin digestion, and analyzed by Matrix Assisted Laser Desorption/Ionization Time-Of-Flight/Time-Of-Flight (MALDI-TOF/TOF) mass spectrometry (MS). Tryptic fragment sequences determined by MS were searched against the recently completed S. scabiei annotated genome, leading to the identification of >150 proteins. Only 10 proteins hit to previously identified scabies proteins including actin, tropomyosin, and several ABC transporters. Thirteen proteins had homology to dust mite allergens (members of groups 8, 10, 13, 17, 20, 25, and 28). Most other sequences showed some homology to proteins in other mites and ticks including homologs of calmodulin, calreticulin, lipocalin, and glutathione-S-transferase. These data will now allow the identification of the proteins to which scabies patients produce antibodies, including those that may be good candidates for inclusion in a diagnostic test and vaccine.

Draft genome of the scabies mite.

BACKGROUND: The disease scabies, caused by the ectoparasitic mite, Sarcoptes scabiei, causes significant morbidity in humans and other mammals worldwide. However, there is limited data available regarding the molecular basis of host specificity and host-parasite interactions. Therefore, we sought to produce a draft genome for S. scabiei and use this to identify molecular markers that will be useful for phylogenetic population studies and to identify candidate protein-coding genes that are critical to the unique biology of the parasite. METHODS: S. scabiei var. canis DNA was isolated from living mites and sequenced to ultra-deep coverage using paired-end technology. Sequence reads were assembled into gapped contigs using de Bruijn graph based algorithms. The assembled genome was examined for repetitive elements and gene annotation was performed using ab initio, and homology-based methods. RESULTS: The draft genome assembly was about 56.2 Mb and included a mitochondrial genome contig. The predicted proteome contained 10,644 proteins, ~67 % of which appear to have clear orthologs in other species. The genome also contained more than 140,000 simple sequence repeat loci that may be useful for population-level studies. The mitochondrial genome contained 13 protein coding loci and 20 transfer RNAs. Hundreds of candidate salivary gland protein genes were identified by comparing the scabies mite predicted proteome with sialoproteins and transcripts identified in ticks and other hematophagous arthropods. These include serpins, ferritins, reprolysins, apyrases and new members of the macrophage migration inhibitory factor (MIF) gene family. Numerous other genes coding for salivary proteins, metabolic enzymes, structural proteins, proteins that are potentially immune modulating, and vaccine candidates were identified. The genes encoding cysteine and serine protease paralogs as well as mu-type glutathione S-transferases are represented by gene clusters. S. scabiei possessed homologs for most of the 33 dust mite allergens. CONCLUSION: The draft genome is useful for advancing our understanding of the host-parasite interaction, the biology of the mite and its phylogenetic relationship to other Acari. The identification of antigen-producing genes, candidate immune modulating proteins and pathways, and genes responsible for acaricide resistance offers opportunities for developing new methods for diagnosing, treating and preventing this disease.

A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α.

Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations.

Ligand-regulated heterodimerization of peroxisome proliferator-activated receptor α with liver X receptor α.

Peroxisome proliferator-activated receptor α (PPARα) and liver X receptor α (LXRα) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXRα and PPARα pathways exist, including competition for the same heterodimeric partner, retinoid X receptor α (RXRα). Although data have suggested that PPARα and LXRα may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein-protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPARα and LXRα interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPARα and LXRα response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXRα. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPARα and LXRα in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPARα alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors.

Amelioration of Cryptosporidium parvum infection in vitro and in vivo by targeting parasite fatty acyl-coenzyme A synthetases.

BACKGROUND: Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target. METHODS: A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo. RESULTS: Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8-15 mg/kg/d for 1 week. CONCLUSIONS: The findings of this study not only validated Cryptosporidium ACS (and related acyl-[acyl-carrier-protein]-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients.

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities.

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation.

A rearrangement of the Z chromosome topology influences the sex-linked gene display in the European corn borer, Ostrinia nubilalis.

Males are homogametic (ZZ) and females are heterogametic (WZ) with respect to the sex chromosomes in many species of butterflies and moths (insect order Lepidoptera). Genes on the Z chromosome influence traits involved in larval development, environmental adaptation, and reproductive isolation. To facilitate the investigation of these traits across Lepidoptera, we developed 43 degenerate primer pairs to PCR amplify orthologs of 43 Bombyx mori Z chromosome-linked genes. Of the 34 orthologs that amplified by PCR in Ostrinia nubilalis, 6 co-segregated with the Z chromosome anchor markers kettin (ket) and lactate dehydrogenase (ldh), and produced a consensus genetic linkage map of ~89 cM in combination with 5 AFLP markers. The O. nubilalis and B. mori Z chromosomes are comparatively co-linear, although potential gene inversions alter terminal gene orders and a translocation event disrupted synteny at one chromosome end. Compared to B. mori orthologs, O. nubilalis Z chromosome-linked genes showed conservation of tissue-specific and growth-stage-specific expression, although some genes exhibited species-specific expression across developmental stages or tissues. The O. nubilalis Z chromosome linkage map provides new tools for isolating quantitative trait loci (QTL) involved in sex-linked traits that drive speciation and it exposes genome rearrangements as a possible mechanism for differential gene regulation in Lepidoptera.

The apicomplexan Cryptosporidium parvum possesses a single mitochondrial-type ferredoxin and ferredoxin:NADP+ reductase system.

We have successfully expressed recombinant mitochondrial-type ferredoxin (mtFd) and ferredoxin:NADP(+) reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time for an apicomplexan. Both C. parvum mtFd (CpmtFd) and FNR (CpmtFNR) were obtained and purified as holo-proteins, in which the correct assembly of [2Fe-2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/vis and electron paramagnetic resonance. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c-coupled assay that followed a typical Michaelis-Menten kinetics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals and could be explored as potential drug targets in Cryptosporidium and other apicomplexans.

An apicomplexan ankyrin-repeat histone deacetylase with relatives in photosynthetic eukaryotes.

Cryptosporidium parvum is a member of the Apicomplexa that lacks a plastid and associated nuclear-encoded genes, which has hampered its use in evolutionary comparisons with algae and eliminated a pool of potentially useful drug targets. Here we show that apicomplexan parasites possess an unusual family of class II histone deacetylase (HDAC) proteins with orthologues that are present in other chromalveolates and primitive algae. A striking feature of these HDAC proteins is the presence of ankyrin repeats in the amino-terminus that appear to be required for enzyme activity. In vitro and in vivo analyses of the C. parvum orthologue indicate that this subclass of chromatin-remodelling proteins is targeted by the anti-cancer drug suberoylanilide hydroxamic acid and that these proteins are most likely involved in the essential process of H4 histone deacetylation that coincides with DNA replication. We propose that members of this novel class of histone deacetylase can serve as promising new targets for treatments against debilitating diseases such as cryptosporidosis, toxoplasmosis and malaria.