ABSTRACT
The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.
Subject(s)
Mammary Glands, Animal/growth & development , Period Circadian Proteins/metabolism , Animals , Circadian Rhythm/genetics , Epithelial Cells/metabolism , Female , Humans , Immunohistochemistry , Mammary Glands, Animal/metabolism , Mice , Organogenesis , Real-Time Polymerase Chain ReactionABSTRACT
Landscape heterogeneity can shape genetic structure and functional connectivity of populations. When this heterogeneity imposes variable costs of moving across the landscape, populations can be structured according to a pattern of "isolation by resistance" (IBR). At the same time, divergent local environmental filters can limit gene flow, creating an alternative pattern of "isolation by environment" (IBE). Here, we evaluate IBR and IBE in the insect-pollinated, biennial plant Sabatia angularis (L.) Pursh (Gentianaceae) across serpentine grasslands in the fragmented landscape of SE Pennsylvania, USA using ~4500 neutral SNP loci. Specifically, we test the extent to which radical alteration of the landscape matrix by humans has fundamentally altered the cost of movement, imprinting a pattern of IBR dictated by land use type and intensity, and the potential for IBE in relation to a gradient of heavy metal concentrations found in serpentine soil. We reveal a strong signal of IBR and a weak signal of IBE across sites, indicating the greater importance of the landscape matrix in shaping genetic structure of S. angularis populations in the study region. Based on Circuitscape and least cost path approaches, we find that both low- and high-intensity urbanization resist gene flow by orders of magnitude greater than "natural" habitats, although resistance to low-intensity urbanization weakens at larger spatial scales. While cropland presents a substantially lower barrier than urban development, cumulative human land use surrounding populations predicts within-population genetic diversity and inbreeding in S. angularis. Our results emphasize the role of forest buffers and corridors in facilitating gene flow between serpentine grassland patches and averting local extinction of plant populations.
Subject(s)
Gene Flow , Gentianaceae/genetics , Grassland , Ecosystem , Genetic Structures , PennsylvaniaABSTRACT
The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor-induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.
Subject(s)
Benzo(a)pyrene/metabolism , Breast/cytology , Circadian Rhythm , DNA Adducts/metabolism , Mammary Glands, Animal/cytology , Animals , Biomarkers/metabolism , Breast/metabolism , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Cell Line , Circadian Rhythm/genetics , Cytochrome P-450 CYP1A1/metabolism , Female , Gene Expression Regulation , Humans , Mice , Models, BiologicalABSTRACT
In Marfan Syndrome (MFS), development of thoracic aortic aneurysms (TAAs) is characterized by degeneration of the medial layer of the aorta, including fragmentation and loss of elastic fibers, phenotypic changes in the smooth muscle cells, and an increase in the active form of transforming growth factor-ß (TGFß), which is thought to play a major role in development and progression of the aneurysm. We hypothesized that regional difference in elastic fiber fragmentation contributes to TGFß activation and hence the localization of aneurysm formation. The fibrillin-1-deficient mgR/mgR mouse model of MFS was used to investigate regional changes in elastin fiber fragmentation, TGFß activation and changes in gene expression as compared to wild-type littermates. Knockdown of Smad 2 and Smad 3 with shRNA was used to determine the role of the specific transcription factors in gene regulation in aortic smooth muscle cells. We show increased elastin fiber fragmentation in the regions associated with aneurysm formation and altered TGFß signaling in these regions. Differential effects of Smad 2 and Smad 3 were observed in cultured smooth muscle cells by shRNA-mediated knockdown of expression of these transcription factors. Differential signaling through Smad 2 and Smad 3 in regions of active vascular remodeling likely contribute to aneurysm formation in the mgR/mgR model of MFS. Increased elastin fiber fragmentation in these regions is associated with these changes as compared to other regions of the thoracic aorta and may contribute to the changes in TGFß signaling in these regions.
Subject(s)
Aorta/metabolism , Aorta/pathology , Elastic Tissue/pathology , Marfan Syndrome/metabolism , Marfan Syndrome/pathology , Organ Specificity , Transforming Growth Factor beta/metabolism , Animals , Aorta, Thoracic/metabolism , Aorta, Thoracic/pathology , Aortic Aneurysm, Thoracic/genetics , Aortic Aneurysm, Thoracic/pathology , Disease Models, Animal , Disease Progression , Elastic Tissue/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Mice, 129 Strain , Mice, Inbred C57BL , Phosphorylation , RNA, Small Interfering/metabolism , Signal Transduction , Smad2 Protein/metabolism , Smad3 Protein/metabolismABSTRACT
We have previously demonstrated that the bHLH/PAS transcription factor, singleminded 2s (Sim2s), is required for proper mammary ductal morphogenesis and luminal epithelial differentiation. Furthermore, loss of Sim2s in breast cancer cells resulted in downregulation of epithelial markers and acquisition of a basal-like phenotype. The objective of this study was to further define the role of Sim2s in mammary differentiation. We found that Sim2s is developmentally regulated throughout mammary gland development with highest expression during lactation. Mammary glands from nulliparous mice expressing Sim2s driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter were morphologically indistinguishable from wild-type mice but displayed hallmarks of precocious lactogenic differentiation. These included elevated expression of the milk protein genes Wap and Csn2, and apical localization of the lactation marker Npt2b. Consistent with the in vivo results, Sim2s enhanced prolactin-mediated Csn2 expression in HC11 and CIT3 mouse mammary epithelial cells, and downregulation of Sim2s by shRNA in HC11 cells inhibited Csn2 expression. Chromatin immunoprecipitation (ChIP) analyses of the Csn2 gene found that Sim2s associates with the Csn2 promoter and re-ChIP experiments showed that Sim2s interacted with the RNA II polymerase (RNAPII) complex. Together, these data demonstrate, for the first time, that Sim2s is required for establishing and maintaining mammary gland differentiation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Cell Differentiation/genetics , Lactation/genetics , Mammary Glands, Animal/growth & development , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/physiology , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/physiology , Female , Gene Expression Regulation, Developmental , Lactation/physiology , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/physiology , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Haematobia irritans irritans (Linnaeus, 1758: Diptera: Muscidae), the horn fly, is an external parasite of penned and pastured livestock that causes a major economic impact on cattle production worldwide. Pesticides such as synthetic pyrethroids and organophosphates are routinely used to control horn flies; however, resistance to these chemicals has become a concern in several countries. To further elucidate the molecular mechanisms of resistance in horn fly populations, we sequenced the transcriptomes of ten populations of horn flies from the southern US possessing varying degrees of pesticide resistance levels to pyrethroids, organophosphates, and endosulfans. We employed an Illumina paired end HiSeq approach, followed by de novo assembly of the transcriptomes using CLC Genomics Workbench 8.0.1 De Novo Assembler using multiple kmers, and annotation using Blast2GO PRO version 5.2.5. The Gene Ontology biological process term Response to Insecticide was found in all the populations, but at an increased frequency in the populations with higher levels of insecticide resistance. The raw sequence reads are archived in the Sequence Read Archive (SRA) and assembled population transcriptomes in the Transcriptome Shotgun Assembly (TSA) at the National Center for Biotechnology Information (NCBI).
ABSTRACT
Knowledge of host associations of blood-feeding vectors may afford insights into managing disease systems and protecting public health. However, the ability of methods to distinguish bloodmeal sources varies widely. We used two methods-Sanger sequencing and amplicon deep sequencing-to target a 228 bp region of the vertebrate Cytochrome b gene and determine hosts fed upon by triatomines (n = 115) collected primarily in Texas, USA. Direct Sanger sequencing of PCR amplicons was successful for 36 samples (31%). Sanger sequencing revealed 15 distinct host species, which included humans, domestic animals (Canis lupus familiaris, Ovis aries, Gallus gallus, Bos taurus, Felis catus, and Capra hircus), wildlife (Rattus rattus, Incilius nebulifer, Sciurus carolinensis, Sciurus niger, and Odocoileus virginianus), and captive animals (Panthera tigris, Colobus spp., and Chelonoidis carbonaria). Samples sequenced by the Sanger method were also subjected to Illumina MiSeq amplicon deep sequencing. The amplicon deep sequencing results (average of 302,080 usable reads per sample) replicated the host community revealed using Sanger sequencing, and detected additional hosts in five triatomines (13.9%), including two additional blood sources (Procyon lotor and Bassariscus astutus). Up to four bloodmeal sources were detected in a single triatomine (I. nebulifer, Homo sapiens, C. lupus familiaris, and S. carolinensis). Enhanced understanding of vector-host-parasite networks may allow for integrated vector management programs focusing on highly-utilized and highly-infected host species.
Subject(s)
Chagas Disease , Deer , Trypanosoma cruzi , Animals , Animals, Domestic/genetics , Cats , Cattle , Chagas Disease/parasitology , Deer/genetics , Dogs , High-Throughput Nucleotide Sequencing , Trypanosoma cruzi/geneticsABSTRACT
Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of cyclic stretch. Quantitative real-time PCR was used to measure changes in mRNA expression of a profile of genes representing the major smooth muscle phenotypes. Genes associated with the contractile state were both upregulated (e.g., calponin) and downregulated (e.g., α-2-actin), and genes associated with the synthetic state were likewise both upregulated (e.g., SKI-like oncogene) and downregulated (e.g., collagen III). In addition, cells aligned with an orientation perpendicular to the maximal stretch direction. We have developed an in vitro cell culture device that can produce non-uniform stretch environments similar to in vivo mechanics. Cells stretched with this device showed alignment and altered mRNA expression indicative of phenotype modulation. Understanding these processes as they relate to in vivo pathologies could enable a more accurately targeted treatment to heal or inhibit disease, either through implantable device design or pharmaceutical approaches.
Subject(s)
Fibroblasts/cytology , Gene Expression/physiology , Muscle Stretching Exercises/methods , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , 3T3 Cells , Animals , Biomechanical Phenomena/physiology , Cell Membrane/physiology , Cells, Cultured , Equipment Design , Fibroblasts/physiology , Mechanotransduction, Cellular/physiology , Mice , Physical Stimulation/methods , Stress, Mechanical , Surface PropertiesABSTRACT
Ticks from the genus Rhipicephalus have enormous global economic impact as ectoparasites of cattle. Rhipicephalus microplus and Rhipicephalus annulatus are known to harbor infectious pathogens such as Babesia bovis, Babesia bigemina, and Anaplasma marginale. Having reference quality genomes of these ticks would advance research to identify druggable targets for chemical entities with acaricidal activity and refine anti-tick vaccine approaches. We sequenced and assembled the genomes of R. microplus and R. annulatus, using Pacific Biosciences and HiSeq 4000 technologies on very high molecular weight genomic DNA. We used 22 and 29 SMRT cells on the Pacific Biosciences Sequel for R. microplus and R. annulatus, respectively, and 3 lanes of the Illumina HiSeq 4000 platform for each tick. The PacBio sequence yields for R. microplus and R. annulatus were 21.0 and 27.9 million subreads, respectively, which were assembled with Canu v. 1.7. The final Canu assemblies consisted of 92,167 and 57,796 contigs with an average contig length of 39,249 and 69,055 bp for R. microplus and R. annulatus, respectively. Annotated genome quality was assessed by BUSCO analysis to provide quantitative measures for each assembled genome. Over 82% and 92% of the 1066 member BUSCO gene set was found in the assembled genomes of R. microplus and R. annulatus, respectively. For R. microplus, only 189 of the 1066 BUSCO genes were missing and only 140 were present in a fragmented condition. For R. annulatus, only 75 of the BUSCO genes were missing and only 109 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.
ABSTRACT
Quantitative trait loci (QTL) analysis could help to identify suitable molecular markers for marker-assisted breeding (MAB). A mapping population of 124 F5:7recombinant inbred lines derived from the cross 'TAM 112'/'TAM 111' was grown under 28 diverse environments and evaluated for grain yield, test weight, heading date, and plant height. The objective of this study was to detect QTL conferring grain yield and agronomic traits from multiple mega-environments. Through a linkage map with 5,948 single nucleotide polymorphisms (SNPs), 51 QTL were consistently identified in two or more environments or analyses. Ten QTL linked to two or more traits were also identified on chromosomes 1A, 1D, 4B, 4D, 6A, 7B, and 7D. Those QTL explained up to 13.3% of additive phenotypic variations with the additive logarithm of odds (LOD(A)) scores up to 11.2. The additive effect increased yield up to 8.16 and 6.57 g m-2 and increased test weight by 2.14 and 3.47 kg m-3 with favorable alleles from TAM 111 and TAM 112, respectively. Seven major QTL for yield and six for TW with one in common were of our interest on MAB as they explained 5% or more phenotypic variations through additive effects. This study confirmed previously identified loci and identified new QTL and the favorable alleles for improving grain yield and agronomic traits.
ABSTRACT
To better understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant lineage distribution in a college campus population, we carried out viral genome surveillance over a 7-week period from January to March 2021. Among the sequences were three novel viral variants: BV-1 with a B.1.1.7/20I genetic background and an additional spike mutation Q493R, associated with a mild but longer-than-usual COVID-19 case in a college-age person, BV-2 with a T478K mutation on a 20B genetic background, and BV-3, an apparent recombinant lineage. This work highlights the potential of an undervaccinated younger population as a reservoir for the spread and generation of novel variants. This also demonstrates the value of whole genome sequencing as a routine disease surveillance tool.
Subject(s)
COVID-19/virology , Disease Reservoirs/virology , Mutation , SARS-CoV-2/genetics , Students/statistics & numerical data , Universities , Adult , COVID-19/etiology , Genome, Viral , Humans , Neutralization Tests , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Young AdultABSTRACT
The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable ([Formula: see text] among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a large-effect risk locus (P-value < 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE ≥ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. white-tailed deer produced mean genomic prediction accuracies ≥ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.
Subject(s)
Deer , Prions , Wasting Disease, Chronic , Animals , Deer/genetics , Genome-Wide Association Study , Genomics , Prions/genetics , Wasting Disease, Chronic/geneticsABSTRACT
BACKGROUND: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function. RESULTS: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. CONCLUSIONS: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.
Subject(s)
DNA Polymerase II/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Factors, General/metabolism , Transcription Initiation Site , Transcription Initiation, Genetic , Models, Genetic , Promoter Regions, Genetic , Saccharomyces cerevisiae/geneticsABSTRACT
The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 (Per1(ldc) and Per1(ldc)/Per2(ldc)). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/drug effects , Carcinogens/toxicity , Cell Cycle Proteins/metabolism , Liver/drug effects , Nuclear Proteins/metabolism , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/drug effects , Signal Transduction/drug effects , Transcription Factors/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Basic Helix-Loop-Helix Transcription Factors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1B1 , Enzyme Induction , Female , Liver/enzymology , Liver/metabolism , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Period Circadian Proteins , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Transcription Factors/geneticsABSTRACT
DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non-model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field-collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double-Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7-13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre-sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower-quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.
ABSTRACT
The longhorned tick, Haemaphysalis longicornis, feeds upon a wide range of bird and mammalian hosts. Mammalian hosts include cattle, deer, sheep, goats, humans, and horses. This tick is known to transmit a number of pathogens causing tick-borne diseases, and was the vector of a recent serious outbreak of oriental theileriosis in New Zealand. A New Zealand-USA consortium was established to sequence, assemble, and annotate the genome of this tick, using ticks obtained from New Zealand's North Island. In New Zealand, the tick is considered exclusively parthenogenetic and this trait was deemed useful for genome assembly. Very high molecular weight genomic DNA was sequenced on the Illumina HiSeq4000 and the long-read Pac Bio Sequel platforms. Twenty-eight SMRT cells produced a total of 21.3 million reads which were assembled with Canu on a reserved supercomputer node with access to 12 TB of RAM, running continuously for over 24 days. The final assembly dataset consisted of 34,211 contigs with an average contig length of 215,205 bp. The quality of the annotated genome was assessed by BUSCO analysis, an approach that provides quantitative measures for the quality of an assembled genome. Over 95% of the BUSCO gene set was found in the assembled genome. Only 48 of the 1066 BUSCO genes were missing and only 9 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.
ABSTRACT
Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility.
Subject(s)
Evolution, Molecular , Fertility/genetics , Horses/genetics , Y Chromosome/genetics , Animals , Ascaridoidea/genetics , Equidae/genetics , Gene Dosage/genetics , Gene Transfer, Horizontal , Hybridization, Genetic , Male , Phylogeny , Testis/metabolism , X Chromosome/geneticsABSTRACT
Northern bobwhite (Colinus virginianus; hereafter bobwhite) and scaled quail (Callipepla squamata) populations have suffered precipitous declines across most of their US ranges. Illumina-based first- (v1.0) and second- (v2.0) generation draft genome assemblies for the scaled quail and the bobwhite produced N50 scaffold sizes of 1.035 and 2.042 Mb, thereby producing a 45-fold improvement in contiguity over the existing bobwhite assembly, and ≥90% of the assembled genomes were captured within 1313 and 8990 scaffolds, respectively. The scaled quail assembly (v1.0 = 1.045 Gb) was â¼20% smaller than the bobwhite (v2.0 = 1.254 Gb), which was supported by kmer-based estimates of genome size. Nevertheless, estimates of GC content (41.72%; 42.66%), genome-wide repetitive content (10.40%; 10.43%), and MAKER-predicted protein coding genes (17,131; 17,165) were similar for the scaled quail (v1.0) and bobwhite (v2.0) assemblies, respectively. BUSCO analyses utilizing 3023 single-copy orthologs revealed a high level of assembly completeness for the scaled quail (v1.0; 84.8%) and the bobwhite (v2.0; 82.5%), as verified by comparison with well-established avian genomes. We also detected 273 putative segmental duplications in the scaled quail genome (v1.0), and 711 in the bobwhite genome (v2.0), including some that were shared among both species. Autosomal variant prediction revealed â¼2.48 and 4.17 heterozygous variants per kilobase within the scaled quail (v1.0) and bobwhite (v2.0) genomes, respectively, and estimates of historic effective population size were uniformly higher for the bobwhite across all time points in a coalescent model. However, large-scale declines were predicted for both species beginning â¼15-20 KYA.
Subject(s)
Colinus/genetics , Evolution, Molecular , Genetic Variation , Genome , Genomics , Quail/genetics , Animals , Computational Biology/methods , DNA Copy Number Variations , Databases, Nucleic Acid , Gene Duplication , Genomics/methods , Molecular Sequence Annotation , Polymorphism, Single Nucleotide , Population Density , Sequence Deletion , Whole Genome SequencingABSTRACT
Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.
Subject(s)
Breast Neoplasms/metabolism , Estradiol/physiology , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic , Sp Transcription Factors/metabolism , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Base Sequence , Cell Line, Tumor , Cell Nucleus/metabolism , Estradiol/metabolism , Humans , Molecular Sequence Data , Neovascularization, Pathologic , Promoter Regions, GeneticABSTRACT
Herein, we evaluated the concordance of population inferences and conclusions resulting from the analysis of short mitochondrial fragments (i.e., partial or complete D-Loop nucleotide sequences) versus complete mitogenome sequences for 53 bobwhites representing six ecoregions across TX and OK (USA). Median joining (MJ) haplotype networks demonstrated that analyses performed using small mitochondrial fragments were insufficient for estimating the true (i.e., complete) mitogenome haplotype structure, corresponding levels of divergence, and maternal population history of our samples. Notably, discordant demographic inferences were observed when mismatch distributions of partial (i.e., partial D-Loop) versus complete mitogenome sequences were compared, with the reduction in mitochondrial genomic information content observed to encourage spurious inferences in our samples. A probabilistic approach to variant prediction for the complete bobwhite mitogenomes revealed 344 segregating sites corresponding to 347 total mutations, including 49 putative nonsynonymous single nucleotide variants (SNVs) distributed across 12 protein coding genes. Evidence of gross heteroplasmy was observed for 13 bobwhites, with 10 of the 13 heteroplasmies involving one moderate to high frequency SNV. Haplotype network and phylogenetic analyses for the complete bobwhite mitogenome sequences revealed two divergent maternal lineages (dXY = 0.00731; FST = 0.849; P < 0.05), thereby supporting the potential for two putative subspecies. However, the diverged lineage (n = 103 variants) almost exclusively involved bobwhites geographically classified as Colinus virginianus texanus, which is discordant with the expectations of previous geographic subspecies designations. Tests of adaptive evolution for functional divergence (MKT), frequency distribution tests (D, FS) and phylogenetic analyses (RAxML) provide no evidence for positive selection or hybridization with the sympatric scaled quail (Callipepla squamata) as being explanatory factors for the two bobwhite maternal lineages observed. Instead, our analyses support the supposition that two diverged maternal lineages have survived from pre-expansion to post-expansion population(s), with the segregation of some slightly deleterious nonsynonymous mutations.