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Genetic variants of mitochondrial DNA at the individual (heteroplasmy) and population (polymorphism) levels provide insight into their roles in multiple cellular and evolutionary processes. However, owing to the paucity of genome-wide data at the within-individual and population levels, the broad patterns of these two forms of variation remain poorly understood. Here, we analyze 1,804 complete mitochondrial genome sequences from Daphnia pulex, Daphnia pulicaria, and Daphnia obtusa. Extensive heteroplasmy is observed in D. obtusa, where the high level of intraclonal divergence must have resulted from a biparental-inheritance event, and recombination in the mitochondrial genome is apparent, although perhaps not widespread. Global samples of D. pulex reveal remarkably low mitochondrial effective population sizes

Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5′ ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and reaction cleanups, a STRIPE-seq library can be constructed in about 5 h. We show application of STRIPE-seq to TSS profiling in yeast and human cells and show that it can also be effectively used for quantification of transcript levels and analysis of differential gene expression. In conjunction with our ready-to-use computational workflows, STRIPE-seq is a straightforward, efficient means by which to probe the landscape of transcriptional initiation.

Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility., Bacterial TAL effectors hijack host gene expression to mediate disease susceptibility, and host plants counteract TAL effectors with resistance genes to thwart pathogen infection. This study reports the cloning of Xa7, demonstrates its induction by the essential virulence TAL effectors AvrXa7 and PthXo3, and documents its prevalence in indica rice cultivars.

Comparing genomes of closely related genotypes from populations with distinct demographic histories can help reveal the impact of effective population size on genome evolution. For this purpose, we present a high quality genome assembly of Daphnia pulex (PA42), and compare this with the first sequenced genome of this species (TCO), which was derived from an isolate from a population with >90% reduction in nucleotide diversity. PA42 has numerous similarities to TCO at the gene level, with an average amino acid sequence identity of 98.8 and >60% of orthologous proteins identical. Nonetheless, there is a highly elevated number of genes in the TCO genome annotation, with ∼7000 excess genes appearing to be false positives. This view is supported by the high GC content, lack of introns, and short length of these suspicious gene annotations. Consistent with the view that reduced effective population size can facilitate the accumulation of slightly deleterious genomic features, we observe more proliferation of transposable elements (TEs) and a higher frequency of gained introns in the TCO genome.

Large-scale transcription start site (TSS) profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis-regulatory landscape of the water flea Daphnia pulex, an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed Cap Analysis of Gene Expression (CAGE) with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a “Daphnia Promoter Atlas,” i.e., a catalog of active promoters across the surveyed states. Analysis of the distribution of promoters revealed evidence for widespread alternative promoter usage in D. pulex, in addition to a prominent fraction of compactly-arranged promoters in divergent orientations. We carried out de novo motif discovery using CAGE-defined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements (e.g., TATA and Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable state-specific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis-regulatory regions in metazoans, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

Application of Transcription Start Site (TSS) profiling technologies, coupled with large-scale next-generation sequencing (NGS) has yielded valuable insights into the location, structure, and activity of promoters across diverse metazoan model systems. In insects, TSS profiling has been used to characterize the promoter architecture of Drosophila melanogaster (Hoskins et al., Genome Res 21(2):182-192, 2011) and subsequently was employed to reveal widespread transposon-driven alternative promoter usage in the fruit fly (Batut et al., Genome Res 23:169-180, 2012).In this chapter we discuss the computational analysis of the experimental data derived from one of TSS profiling methods, RAMPAGE (RNA Annotation and Mapping of Promoters for Analysis of Gene Expression) that can be used for the precise, quantitative identification of promoters in insect genomes. We demonstrate this using the software tools GoRAMPAGE (Brendel and Raborn, GoRAMPAGE-A workflow for promoter detection by 5'-read mapping. https://github.com/BrendelGroup/GoRAMPAGE , 2016) and TSRchitect (Raborn and Brendel, TSRchitect: promoter identification from large-scale TSS profiling data. R Bioconductor package version 1.8.0 [Online]. Available: http://bioconductor.org/packages/release/bioc/html/TSRchitect.html , 2017), providing detailed instructions with the aim of taking the user from raw reads to processed results.

Application of Transcription Start Site (TSS) profiling technologies, coupled with large-scale next-generation sequencing (NGS) has yielded valuable insights into the location, structure, and activity of promoters across diverse metazoan model systems. In insects, TSS profiling has been used to characterize the promoter architecture of Drosophila melanogaster (Hoskins et al., Genome Res 21(2):182-192, 2011) and subsequently was employed to reveal widespread transposon-driven alternative promoter usage in the fruit fly (Batut et al., Genome Res 23:169-180, 2012).In this chapter we discuss the computational analysis of the experimental data derived from one of TSS profiling methods, RAMPAGE (RNA Annotation and Mapping of Promoters for Analysis of Gene Expression) that can be used for the precise, quantitative identification of promoters in insect genomes. We demonstrate this using the software tools GoRAMPAGE (Brendel and Raborn, GoRAMPAGE-A workflow for promoter detection by 5'-read mapping. https://github.com/BrendelGroup/GoRAMPAGE , 2016) and TSRchitect (Raborn and Brendel, TSRchitect: promoter identification from large-scale TSS profiling data. R Bioconductor package version 1.8.0 [Online]. Available: http://bioconductor.org/packages/release/bioc/html/TSRchitect.html , 2017), providing detailed instructions with the aim of taking the user from raw reads to processed results.

Large-scale transcription start site (TSS) profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis -regulatory landscape of the water flea Daphnia pulex , an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed Cap Analysis of Gene Expression (CAGE) with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a 'Daphnia Promoter Atlas'- a catalog of active promoters across the surveyed states. Analysis of the distribution of promoters revealed evidence for widespread alternative promoter usage in D. pulex , in addition to a prominent fraction of compactly-arranged promoters in divergent orientations. We carried out de novo motif discovery using CAGE-defined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements ( e.g. TATA, Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable state-specific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis -regulatory regions in arthropods, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) differ functionally in that hENT2 generally displays lower affinity for its nucleoside permeants and is less sensitive to inhibition by the coronary vasodilators dilazep and dipyridamole. In previous work, we demonstrated that mutation of residues 33 (Met versus Ile) of hENT1 and hENT2 altered sensitivity to dilazep and dipyridamole and that the hENT2 mutant (I33M) displayed a K(m) value for uridine that was lower than that of hENT2 and similar to that of hENT1 (J Biol Chem 277:395-401, 2002). In this study, we report results of an in-depth investigation of the role of residue 33 in hENT2. We found that hENT2-I33M displayed decreased K(m) values for both pyrimidine and purine nucleosides and increased V(max) values for purine nucleosides. Cys or Ser at position 33 had similar effects on the kinetic parameters of hENT2 as Met, indicating that hydrophobic (Met and Cys) or hydrogen-bonding energy (Ser) contributed to permeant binding by these residues. hENT2-I33M and I33C displayed increased sensitivities to dipyridamole compared with wild-type hENT2, hENT2-I33A, and hENT2-I33S, suggesting interaction of the sulfur atom of Met and Cys with aromatic moieties on dipyridamole. hENT2-I33C was inhibited by the membrane-impermeant sulfhydryl reactive reagent p-chloromercuribenzyl sulfonate, and uridine, adenosine, and dipyridamole protected against inhibition. Our results indicated that residue 33 resides in an extracellular domain as predicted by the current hENT2 topology model and suggested that it is a functionally important component of both the permeant and dipyridamole binding sites.

The human equilibrative nucleoside transporters 1 and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants. The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis. Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases. To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of [3H]uridine by recombinant hENT1 and hENT2 produced in yeast. hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters. hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base.Key words: hENT2, hENT1, araC, uridine, NBMPR.