Draft sequencing and assembly of the genome of the world's largest fish, the whale shark: Rhincodon typus Smith 1828.

BACKGROUND: The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. RESULTS: We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. CONCLUSIONS: This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

Direct amplification, sequencing and profiling of Chlamydia trachomatis strains in single and mixed infection clinical samples.

Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1-1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.

Cardiac-targeted transgenic mutant mitochondrial enzymes: mtDNA defects, antiretroviral toxicity and cardiomyopathy.

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (Pol gamma) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol gamma and mtDNA replication. Cardiac "dominant negative" murine transgenes (TGs; Pol gamma Y955C, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in "2 x 2" studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs ( approximately 50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass ( approximately 50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy.

Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma.

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol gamma hypothesis.

DESIGN: Nucleoside reverse transcriptase inhibitors (NRTIs) exhibit mitochondrial toxicity. The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium served to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro. METHODS: Zidovudine (3'-azido-2',3'-deoxythymidine; ZDV), stavudine (2', 3'-didehydro-2', 3'-deoxythymidine; d4T), or lamivudine ((-)-2'-deoxy-3'-thiacytidine; 3TC) were administered individually to TGs and wild-type (WT) littermates (35 days) at human doses with drug-free vehicle as control. Left ventricle (LV) mass was defined echocardiographically, mitochondrial ultrastructural defects were identified by electron microscopy, the abundance of cardiac mtDNA was quantified by real time polymerase chain reaction, and mtDNA-encoded polypeptides were quantified. RESULTS: Untreated TGs exhibited normal LV mass with minor mitochondrial damage. NRTI monotherapy (either d4T or ZDV) increased LV mass in TGs and caused significant mitochondrial destruction. Cardiac mtDNA was depleted in ZDV and d4T-treated TG hearts and mtDNA-encoded polypeptides decreased. Changes were absent in 3TC-treated cohorts. In supportive structural observations from molecular modeling, ZDV demonstrated close contacts with K947 and Y951 in the DNA pol gamma active site that were absent in the HIV reverse transcriptase active site. CONCLUSIONS: NRTIs deplete mtDNA and polypeptides, cause mitochondrial structural and functional defects in vivo, follow inhibition kinetics with DNA pol gamma in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol gamma by specific NRTIs are mechanistically important in mitochondrial toxicity.

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS.

Nucleoside reverse transcriptase inhibitors (NRTIs) are antiretrovirals for AIDS with limiting mitochondrial side effects. The mitochondrial deoxynucleotide carrier (DNC) transports phosphorylated nucleosides for mitochondrial DNA replication and can transport phosphorylated NRTIs into mitochondria. Transgenic mice (TG) that exclusively overexpress DNC in the heart tested DNC's role in mitochondrial dysfunction from NRTIs. Two TG lines were created that overexpressed the human DNC gene in murine myocardium. Cardiac and mitochondrial structure and function were examined by magnetic resonance imaging, echocardiography, electrocardiography, transmission electron microscopy, and plasma lactate. Antiretroviral combinations (HAART) that contained NRTIs (stavudine (2', 3'-didehydro-2', 3'-deoxythymidine or d4T)/lamivudine/indinavir; or zidovudine (3' azido-3'-deoxythymidine or AZT)/lamivudine/indinavir; 35 days) were administered to simulate AIDS therapy. In parallel, a HAART combination without NRTIs (nevirapine/efavirenz/indinavir; 35 days) served as an NRTI-sparing, control regimen. Untreated DNC TGs exhibited normal cardiac function but abnormal mitochondrial ultrastructure. HAART that contained NRTIs caused cardiomyopathy in TGs with increased left ventricle mass and volume, heart rate variability, and worse mitochondrial ultrastructural defects. In contrast, treatment with an NRTI-sparing HAART regimen caused no cardiac changes. Data suggest the DNC is integral to mitochondrial homeostasis in vivo and may relate mechanistically to mitochondrial dysfunction in patients treated with HAART regimens that contain NRTIs.

HIV viral protein R causes atrial cardiomyocyte mitosis, mesenchymal tumor, dysrhythmia, and heart failure.

HIV viral protein R (Vpr) affects the immunocyte cell cycle and circulates as free polypeptide in plasma of AIDS patients. Effects of Vpr on cardiomyocytes were explored using transgenic mice (TG) with Vpr targeted to cardiomyocytes by the alpha-myosin heavy-chain promoter. TG and WT littermate hearts were evaluated histopathologically, ultrastructurally, molecularly via RNA microarray analysis and quantitative RT-PCR, and functionally by cardiac magnetic resonance imaging (MRI) and electrocardiograms (ECG). Six hemizygous lines were created (Vpr(a,b,c,d,e,h)). Vpr RNA was expressed exclusively in myocardium and Vpr mRNA expression correlated with phenotypic changes. Vpr(b) exhibited the highest expression and mortality. TGs developed congestive heart failure ( approximately 8 weeks), abnormal cardiomyocyte nuclei and mitoses ( approximately 12 weeks), and became moribund ( approximately 20 weeks) with atrial mesenchymal tumors. MRI revealed four-chamber dilation, defective contraction, and atrial masses. Pathologically, cardiomegaly and atrial mesenchymal tumors occurred ( approximately 16-20 weeks). ECGs showed prolonged R-R, Q-T, and P-R intervals ( approximately 12 weeks). RNA encoding collagen and bone morphogenic protein 4, 6, and 7 were increased. Vpr targeted to cardiomyocytes caused defective contractility and atrial tumors. Since some Vpr cardiomyocytic effects resemble those found in terminally differentiated immunocytes, some pathogenetic mechanisms may be shared at the subcellular level.

Nucleoside reverse transcriptase inhibitors impair endothelium-dependent relaxation by increasing superoxide.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been used successfully to reduce acquired immunodeficiency syndrome mortality. However, the use of these compounds is associated with numerous tissue toxicities, including cardiomyopathy. These studies address the effects of NRTIs on vascular function. Functional assays of contraction and relaxation were performed on isolated mouse aorta segments obtained from FVB/n mice exposed to zidovudine (AZT), stavudine, or water for 35 days. AZT and stavudine treatment impaired sensitivity to endothelium-dependent relaxation by acetylcholine. Dihydroethidium staining revealed that AZT treatment was associated with an increase in superoxide levels. Pretreatment of AZT-treated vessels with tiron (1 mM), a free radical scavenger, restored endothelium-dependent relaxation in mice. In cellular preparations, electron spin resonance measurements revealed elevated superoxide in cultured endothelial cells exposed to AZT; elevation was dependent on the length of exposure. These results indicate that NRTIs impair endothelium-dependent relaxation by increasing superoxide levels and suggest that NRTI therapy contributes to cardiovascular complications in acquired immunodeficiency syndrome.