The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.
The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as searchable DNA sequence database ( neuromabseq.ucdavis.edu ) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.
Introduction Understanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2 . Methods We performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression. Results Our studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1 - and lws2 -expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments. Discussion We found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH.
Abstract Viruses are a major contributor to oncogenesis, causing 10-15% of human cancers. Molecular pathways involved in malignant transformation are frequently activated by genetic alterations, including but not limited to, somatic mutations, copy number aberrations, structural variants, and oncoviruses. Precision cancer medicine aims to classify tumors by site, histology, and molecular tests to determine an “individualized” profile of cancer alterations. However, clinical tests for these various alterations are sequential, time consuming, and use a lot of material, which is often quite limited (e.g., biopsies). Moreover, tests for the presence of viral sequence are generally performed separately to tests (such as massively parallel sequencing) to detect human genomic alterations. Here we present a hybrid capture and massively parallel sequencing approach to detect viral infection concurrently with targeted genomic analysis, which may decrease assay costs, increase sensitivity and scalability, and detect many types of alterations, thereby providing a more complete tumor genetic profile all from a single sample. We have created a custom hybrid capture probeset for targeted Illumina sequencing to determine whether oncoviruses are present in tissue samples and also determine if the virus has integrated into the host’s genome. We have created both ‘detection’ and ‘integration’ baits for several oncoviruses, including polyomaviruses, human papilloma viruses, Epstein-Barr virus, human cytomegalovirus, Kaposi sarcoma herpesvirus, human T-lymphotropic virus, and hepatitis B virus. To distinguish between different strains of a single virus, strain-specific detection baits were created to bind to variable regions of viral genomes. The integration bait was designed to bind to regions of the viral genomes that are commonly integrated into the human genome. This baitset can also be combined with other capture panels targeting oncogenes to simultaneously determine infection and integration statuses, as well as somatic mutations, copy number and structural variants. To detect virus presence, reads were aligned to a hybrid reference of both the human, and targeted virus genomes. Viral integration status and integration loci were determined by leveraging discordant read pairs that aligned to both the human genome and a viral genome. We have tested our techniques on tissue samples that were infected with either Merkel Cell Polyomavirus or Epstein-Barr virus, as determined using quantitative polymerase chain reaction (qPCR) or immunohistochemistry (IHC) techniques, and have successfully detected these viruses and identified viral integration loci. Overall, this viral hybrid capture probeset provides the ability to simultaneously determine a tissue sample’s infection and viral integration status alongside other somatic genomic analyses, saving both time and sample material. Citation Format: Robert T. Burns, Samuel S. Hunter, Matthew D. Ducar, Aaron R. Thorner, James A. Decaprio, Paul Van Hummelen, Alexander Frieden, Anwesha Nag, Haley A. Coleman, Michael K. Slevin, Andrea Clapp, Samantha D. Drinan, Suzanne R. McShane, Edwin Thai, Priyanka Shivdasani, Joshua Bohannon, Johann Hoeftberger, Reuben Jacobs, Bruce M. Wollison, Neil A. Patel, Monica D. Manam, Phani Davineni, Matthew Meyerson, Laura E. MacConaill. Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2017-562
Abstract Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis. Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged. Citation Format: Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3644.
Abstract Plant recruit diverse microbial communities from the soil biota. Inter-microbial interactions and connectivity in the root microbiome could play essential roles in plant health by promoting resistance to soil-borne pathogens. Yet, understanding these interactions under field conditions is still scarce. Using a strawberry crop model, we characterized the prokaryotic and fungal communities in the rhizosphere and roots of three strawberry cultivars displaying varying resistance degrees to the soil-borne fungal pathogen Macrophomina phaseolina. We tested the hypothesis that resistant cultivars assemble distinct bacterial and fungal communities that foster microbial connectivity and mediate disease resistance. Our results show that the soil-borne pathogen, M. phaseolina , does not perturb the root microbiome of the strawberry cultivars. Microbiome comparative analysis indicated that the highly susceptible cultivar, Sweet Ann, assembles a distinct microbiome that shows reduced network connectivity, whereas more resistant cultivars were enriched in potential beneficial microbes and showed higher network connectivity. Collectively, these results suggest the role of plant genetic traits in the assembly of beneficial microbiome members. Our study reinforces the eminent role of the plant microbiome as trait of selection in breeding programs and stresses further understandings of the genetic and biological mechanisms that mediate microbiome assembly. Uncovering these mechanisms will be key for future plant breeding programs.
Abstract Sex is determined genetically in most fishes, but the gene responsible for sex determination is not known for the vast majority of fish species, including Chinook Salmon Oncorhynchus tshawytscha . The purpose of this study was to characterize a putative sex‐determining gene (“sexually dimorphic on the Y‐chromosome” [ sdY ] gene) in Chinook Salmon and develop a method to test genomic DNA (gDNA) samples for genetic sex assignment. Using next‐generation sequencing and salmonid DNA sequence data from GenBank, the entire genomic organization of Chinook Salmon sdY was described. The corresponding full‐length complementary DNA (cDNA) sequence generated from total RNA was determined by using a combination of genomic‐based primers and “rapid amplification of cDNA ends” (RACE) PCR assays. A phylogenic analysis was conducted by comparing the Chinook Salmon sdY cDNA sequence with sdY sequences (GenBank) from 10 other teleost species. A multisequence alignment was performed, and a phylogenetic tree was inferred from the alignment, providing evidence for sdY identity. Based upon a TaqMan real‐time PCR assay, a genetic test for male sex using sdY was developed and tested on gDNA isolated from phenotypic female and male Chinook Salmon representing populations in Alaska, Idaho, and Washington. The results provide compelling evidence that sdY is the sex‐determining gene in Chinook Salmon. We developed a reliable assay for sdY that can be used for genetic sex assignment in this species. However, discordance between phenotype and genotype was noted in 13 of 107 phenotypic female Chinook Salmon from Alaska and Washington. Several explanations for this discordance are discussed. Received January 14, 2014; accepted November 25, 2014
