Parallel DNA Research Articles

Engineering yellow fluorescent protein probe for visualization of parallel DNA G-quadruplex

Introduction: The formation of G-quadruplex plays a key role in many biological processes. Therefore, visualization of G-quadruplex is highly essential for design of G-quadruplex-targeted small molecules (drugs). Herein, we report on an engineered fluorescent protein probe which was able to distinguish G-quadruplex topologies.
 Methods: The fluorescent protein probe was generated by genetically incorporating yellow fluorescent protein (YFP) to RNA helicase associated with AU-rich element (RHAU) peptide motif.
 Results: This probe could selectively bind and visualize parallel G-quadruplex structure (T95-2T) at high affinity (Kd~130 nM). Visualization of the parallel G-quadruplex by RHAU-YFP could be easily observed in vitro by using normal Gel Doc or the naked eye.
 Conclusion: The YFP probe could be encoded in cells to provide a powerful tool for detection of parallel G-quadruplexes both in vitro and in vivo.

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes

BackgroundPan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.ResultsAnalysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of > 90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.ConclusionsOur microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

Binding of anticancer drug daunomycin to parallel G-quadruplex DNA [d-(TTGGGGT)]4 leads to thermal stabilization: A multispectroscopic investigation

Guanine rich DNA sequences form four stranded G-quadruplex structures found in telomeric DNA and oncogene promoters. Small molecules binding and stabilizing the G-quadruplex disrupt telomere maintenance and gene regulation, thereby limiting the proliferation of cancer cells. The anti-cancer drug daunomycin binds to both G-quadruplex DNA and duplex DNA. We have undertaken a study of interaction of daunomycin to [d-(TTGGGGT)]4 comprising telomeric DNA sequence from Tetrahymena thermophilia to understand the mechanisms of its action. Absorbance, fluorescence and circular dicroism spectra show significant change on interaction with no change in wavelength maxima. The daunomycin dimers present in free state in solution are disrupted on binding. Presence of all sequential short inter proton distance contacts in nuclear magnetic resonance spectra confirm external binding. The observed inter molecular nuclear Overhauser enhancements, changes in chemical shift and molecular docking studies establish well defined binding of daunomycin at two different sites of G-quadruplex DNA. Thermal stabilization of [d-(TTGGGGT)]4 by 10–15 °C upon daunomycin binding is expected to reduce access of telomerase to its functional site at telomeres. The present studies on mode of action pave the way for alternate derivatives/analogues by chemical modification of anthracyclines to arrive at a more potent telomerase inhibitor.

Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study.

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.

INVITED] Electrophoretic plasmonic nanopore biochip genome sequencer

Our research group is investigating a novel DNA sequencing methodology based on an electrophoretic plasmonic nanopore (EPIGeneS) biochip system used to control the translocation rate of DNA molecules through a fractal array of plasmonic nanopores to enable massively parallel optical recognition DNA sequencing. The EPIGeneS biochip uses nanofluidic conduits to transport geometrically-conformed DNA single molecules though a fractal nanopore “trapping” barrier structure composed of silica nanoparticles positioned within the fluid delivery path of the chip’s nanofluidic channels. Electrophoretic transport of the DNA molecules through the barrier’s nanometer-scaled pores enables the translocation control of DNA single molecules facilitating the Raman spectroscopy detection and measurement of the DNA bases’ spectral signatures as they traverse within the fractal-dimensionality nanopore barrier structures. To enhance the spectroscopy detection and measurement of the DNA nucleobase signatures, the EPIGeneS biochip uses plasmonic structures by coating the surface of the barrier silica nanoparticles with metallic layers to enable the use of surface enhanced Raman spectroscopy imaging detection techniques. This paper describes preliminary results obtained by our group for the development and usage of the EPIGeneS biochip platform for the investigation of the DNA translocation control through silica nanopore barrier structures, and the Raman spectroscopy read-out of DNA base signatures during electrophoretic transport through the fractal nanopore barrier structures.

Novel OTOF gene mutations identified using a massively parallel DNA sequencing technique in DFNB9 deafness

Objectives: This study examined the causative genes in patients with early-onset hearing loss from two Chinese families.Method: Massively parallel sequencing, designed to screen all reported genes associated with hearing loss, was performed in a large number of Chinese individuals with hearing loss. This study enrolled patients with the same OTOF mutation and analyzed their phenotype–genotype correlations.Results: Three novel OTOF mutations (NM_001287489) [c.1550T > C (p.L517P), c.5900_5902delTCA (p.I1967del), and c.4669_4677delCTGACGGTG (p.L1557-V1559del)] were found to be the cause of hearing loss in five patients. In family AH-890, the affected subject homozygous for p.L517P presented with profound hearing loss, while the affected sisters compound heterozygous for p.L517P and p.I1967del had mild-to-moderate hearing loss. The patient with hearing loss in family SD-345 was found to be compound heterozygous for p.L517P and p.L1557-V1559del.Conclusion: Three presumably pathogenic mutations in the OTOF gene were detected for the first time, including the first pathogenic mutation detected in the TM domain. In addition to expanding the spectrum of OTOF mutations resulting in DFNB9, our findings present the diversity of its clinical presentation and indicate that MPS is an efficient approach to identify the causative genes associated with hereditary hearing loss.

Abstract NG06: The clinico-genomics of localized, non-indolent prostate cancer: the CPC-GENE experience

Abstract NG06: The clinico-genomics of localized, non-indolent prostate cancer: the CPC-GENE experience

Differentiating Parallel and Antiparallel DNA Duplexes in the Gas Phase Using Trapped Ion Mobility Spectrometry.

Deoxyribonucleic acids can form a wide variety of structural motifs which differ greatly from the typical antiparallel duplex stabilized by Watson-Crick base pairing. Many of these structures are thought to occur in vivo and may have essential roles in the biology of the cell. Among these is the parallel-stranded duplex-a structural motif in which DNA strands associate in a head-to-head fashion with the 5' ends at the same end of the duplex-which is stabilized by reverse Watson-Crick base pairing. In this study, parallel- and antiparallel-stranded DNA duplexes formed from two different 12-mer oligonucleotides were studied using native electrospray ionization combined with trapped ion mobility spectrometry and mass spectrometry. The DNA duplex charge plays an important role in the gas-phase mobility profile, with a more compact form in negative mode than in positive mode (ΔΩ ≈ 100 Å2 between -4 and +4). Despite sequence mismatches, homo- and hetero-DNA duplexes were formed in solution and transfer to the gas phase, where a more compact structure was observed for the parallel compared to the antiparallel duplexes (ΔΩ ≈ 50 Å2), in good agreement with theoretical calculations. Theoretical studies suggest that a reduction (or compaction) along the helical axis of the parallel and antiparallel DNA duplexes is observed upon transfer to the gas phase.

Microbiome and Culture Based Analysis of Chronic Rhinosinusitis Compared to Healthy Sinus Mucosa.

The role of bacteria in chronic rhinosinusitis (CRS) is still not well understood. Whole microbiome analysis adds new aspects to our current understanding that is mainly based on isolated bacteria. It is still unclear how the results of microbiome analysis and the classical culture based approaches interrelate. To address this, middle meatus swabs and tissue samples were obtained during sinus surgery in 5 patients with CRS with nasal polyps (CRSwNP), 5 patients with diffuse CRS without nasal polyps (CRSsNP), 5 patients with unilateral purulent maxillary CRS (upm CRS) and 3 patients with healthy sinus mucosa. Swabs were cultured, and associated bacteria were identified. Additionally, parts of each tissue sample also underwent culture approaches, and in parallel DNA was extracted for 16S rRNA gene amplicon-based microbiome analysis. From tissue samples 4.2 ± 1.2 distinct species per patient were cultured, from swabs 5.4 ± 1.6. The most frequently cultured species from the swabs were Propionibacterium acnes, Staphylococcus epidermidis, Corynebacterium spp. and Staphylococcus aureus. The 16S-RNA gene analysis revealed no clear differentiation of the bacterial community of healthy compared to CRS samples of unilateral purulent maxillary CRS and CRSwNP. However, the bacterial community of CRSsNP differed significantly from the healthy controls. In the CRSsNP samples Flavobacterium, Pseudomonas, Pedobacter, Porphyromonas, Stenotrophomonas, and Brevundimonas were significantly enriched compared to the healthy controls. Species isolated from culture did not generally correspond with the most abundant genera in microbiome analysis. Only Fusobacteria, Parvimonas, and Prevotella found in 2 unilateral purulent maxillary CRS samples by the cultivation dependent approach were also found in the cultivation independent approach in high abundance, suggesting a classic infectious pathogenesis of odontogenic origin in these two specific cases. Alterations of the bacterial community might be a more crucial factor for the development of CRSsNP compared to CRSwNP. Further studies are needed to investigate the relation between bacterial community characteristics and the development of CRSsNP.

Large Scale Parallel DNA Detection by Two-Dimensional Solid-State Multipore Systems.

We describe a scalable device design of a dense array of multiple nanopores made from nanoscale semiconductor materials to detect and identify translocations of many biomolecules in a massively parallel detection scheme. We use molecular dynamics coupled to nanoscale device simulations to illustrate the ability of this device setup to uniquely identify DNA parallel translocations. We show that the transverse sheet currents along membranes are immune to the crosstalk effects arising from simultaneous translocations of biomolecules through multiple pores, due to their ability to sense only the local potential changes. We also show that electronic sensing across the nanopore membrane offers a higher detection resolution compared to ionic current blocking technique in a multipore setup, irrespective of the irregularities that occur while fabricating the nanopores in a two-dimensional membrane.

TIA: algorithms for development of identity-linked SNP islands for analysis by massively parallel DNA sequencing

BackgroundSingle nucleotide polymorphisms (SNPs) located within the human genome have been shown to have utility as markers of identity in the differentiation of DNA from individual contributors. Massively parallel DNA sequencing (MPS) technologies and human genome SNP databases allow for the design of suites of identity-linked target regions, amenable to sequencing in a multiplexed and massively parallel manner. Therefore, tools are needed for leveraging the genotypic information found within SNP databases for the discovery of genomic targets that can be evaluated on MPS platforms.ResultsThe SNP island target identification algorithm (TIA) was developed as a user-tunable system to leverage SNP information within databases. Using data within the 1000 Genomes Project SNP database, human genome regions were identified that contain globally ubiquitous identity-linked SNPs and that were responsive to targeted resequencing on MPS platforms. Algorithmic filters were used to exclude target regions that did not conform to user-tunable SNP island target characteristics. To validate the accuracy of TIA for discovering these identity-linked SNP islands within the human genome, SNP island target regions were amplified from 70 contributor genomic DNA samples using the polymerase chain reaction. Multiplexed amplicons were sequenced using the Illumina MiSeq platform, and the resulting sequences were analyzed for SNP variations. 166 putative identity-linked SNPs were targeted in the identified genomic regions. Of the 309 SNPs that provided discerning power across individual SNP profiles, 74 previously undefined SNPs were identified during evaluation of targets from individual genomes. Overall, DNA samples of 70 individuals were uniquely identified using a subset of the suite of identity-linked SNP islands.ConclusionsTIA offers a tunable genome search tool for the discovery of targeted genomic regions that are scalable in the population frequency and numbers of SNPs contained within the SNP island regions. It also allows the definition of sequence length and sequence variability of the target region as well as the less variable flanking regions for tailoring to MPS platforms. As shown in this study, TIA can be used to discover identity-linked SNP islands within the human genome, useful for differentiating individuals by targeted resequencing on MPS technologies.

Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus.

In the process of performing their protective functions, neutrophils can form neutrophil extracellular traps (NETs), consisting of DNA in combination with enzymes and histones. The aim of the study was to determine the dynamics of the formation of NETs under the influence of opsonized Staphylococcus aureus and to determine the morphological features of their development in real time by atomic force microscopy. It was found that the maximum formation of NETs was observed after 3hours of co-incubation of neutrophils and opsonized S.aureus. For the first time, the atomic force microscopy method revealed that, at first, large blocks of parallel DNA helices are formed, which then spread in waves, and only then their bifurcation and separation can be observed. Some of the strands formed are covered by a shell, which subsequently completely disappears. Enzymes and histones become clearly visible only after 140 to 150minutes of observation. The DNA helixes move toward the opsonized S.aureus. After NET formation, the cell remains on the substrate only in the form of traces of focal adhesion. This, and the fact that the maximum amount of NETs is formed after 3hours of co-incubation with opsonized S.aureus, suggests that the formation of NETs follows the classical mechanism. The study of the dynamics of formation and the microstructure of NETs makes it possible to estimate the time frame for the implementation of this protective mechanism of the human body when performing the compensatory inflammatory reaction.

Comprehensive analysis of cancers of unknown primary for the biomarkers of response to immune checkpoint blockade therapy

BackgroundCancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Avoiding immune destruction is a major cancer characteristic and therapies aimed at immune checkpoint blockade are in use for several specific cancer types. A comprehensive survey of predictive biomarkers to immune checkpoint blockade in CUP were explored in this study. MethodsAbout 389 cases of CUP were analysed for mutations in 592 genes and 52 gene fusions using a massively parallel DNA sequencing platform (next-generation sequencing [NGS]). Total mutational load (TML) and microsatellite instability (MSI) were calculated from NGS data. PD-L1 expression was explored using immunohistochemistry (with 5% cutoff value). ResultsHigh TML was seen in 11.8% (46/389) of tumours. MSI-high (MSI-H) was detected in 7/384 (1.8%) of tumours. Tumour PD-L1 expression was detected in 80/362 CUP (22%). A small proportion of CUP cases harboured genetic alterations of negative predictive biomarkers to immune checkpoint inhibitors (predictors to hyperprogression) including MDM2 gene amplification (2%) and loss of function JAK2 gene mutations (1%). Amplifications of CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes were also rare (1.4% and 0.8%, respectively). The most frequently mutated genes were TP53 (54%), KRAS (22%), ARID1A (13%), PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%) and PBRM1, STK11, APC, RB1 (5%, respectively). ConclusionsUsing a multiplex testing approach, 28% of CUP carried one or more predictive biomarkers (MSI-H, PD-L1 and/or TML-H) to the immune checkpoint blockade, providing a novel option for treatment in patients with CUP.

Parallel DNA Sequence Approximate Matching with Multi-Length Sequence Aware Approach

Parallel DNA Sequence Approximate Matching with Multi-Length Sequence Aware Approach

WFS1 mutation screening in a large series of Japanese hearing loss patients: Massively parallel DNA sequencing-based analysis.

A heterozygous mutation in the Wolfram syndrome type 1 gene (WFS1) causes autosomal dominant nonsyndromic hereditary hearing loss, DFNA6/14/38, or Wolfram-like syndrome. To date, more than 40 different mutations have been reported to be responsible for DFNA6/14/38. In the present study, WFS1 variants were screened in a large series of Japanese hearing loss (HL) patients to clarify the prevalence and clinical characteristics of DFNA6/14/38 and Wolfram-like syndrome. Massively parallel DNA sequencing of 68 target genes was performed in 2,549 unrelated Japanese HL patients to identify genomic variations responsible for HL. The detailed clinical features in patients with WFS1 variants were collected from medical charts and analyzed. We successfully identified 13 WFS1 variants in 19 probands: eight of the 13 variants were previously reported mutations, including three mutations (p.A684V, p.K836N, and p.E864K) known to cause Wolfram-like syndrome, and five were novel mutations. Variants were detected in 15 probands (2.5%) in 602 families with presumably autosomal dominant or mitochondrial HL, and in four probands (0.7%) in 559 sporadic cases; however, no variants were detected in the other 1,388 probands with autosomal recessive or unknown family history. Among the 30 individuals possessing variants, marked variations were observed in the onset of HL as well as in the presence of progressive HL and tinnitus. Vestibular symptoms, which had been rarely reported, were present in 7 out of 30 (23%) of the affected individuals. The most prevalent audiometric configuration was low-frequency type; however, some individuals had high-frequency HL. Haplotype analysis in three mutations (p.A716T, p.K836T, and p.E864K) suggested that the mutations occurred at these mutation hot spots. The present study provided new insights into the audiovestibular phenotypes in patients with WFS1 mutations.

ScNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

An improved generalized DNA computing model to simulate logic functions and combinational circuits

In this paper a reusable, generalized, parallel DNA computing model is presented to evaluate any logic function at molecular level. The gate strands designed by this algorithm act both as logic operator and sensor to detect the output. Though this model could be employed to simulate vast range of logic functions but for simplicity of explanation theoretical simulation results of DNA based NAND, NOR, half-adder, full-adder and four-bit carry ripple adder are demonstrated to validate this model. The proposed model relies on the induced hairpin formation property of naphthyridine dimer in a G–G mismatched DNA oligo strand which is integrated with a generalized gate design algorithm. Contribution of this work lies in the inclusion of features like single design strategy for any logic function, uniformity in representation of logic 0 and 1 throughout the simulation process and is cost and implementation effective with parallel processing capacity.

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

BackgroundFusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated.FindingsWe generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription.ConclusionOur results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.

Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and Circular Dichroism spectroscopy

Plant derived small molecules, which interact with and stabilize G-quadruplex DNA, act as inhibitors of telomere elongation and oncogene expression in humans. The inhibition of telomerase enzyme has immense potential since it is over expressed in most cancer cells. Interaction of palmatine, an antitumor alkaloid, to parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4, has been investigated using Nuclear Magnetic Resonance (NMR), fluorescence and Circular Dichroism (CD) spectroscopy. Titrations were monitored by 1H and 31P NMR spectra and solution structure of palmatine-[d(TTGGGGT)]4 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by CD, 1H NMR and Differential Scanning Calorimetry (DSC). Binding of palmatine induces 98% enhancement of fluorescence accompanied by blue shift ∼8 nm. CD spectral bands of DNA show minor changes. Diffusion NMR studies confirm formation of a stable complex. Proton NMR signals of palmatine shift upfield upon binding and NOE cross peaks of H10, H3, H28, 5OCH3 protons with T2, A3/G3, G6 and T7 residues reveal dual recognition sites in both G-quadruplex DNA sequences, resulting in thermal stabilization of G-quadruplex by ∼13–17 °C. Restrained molecular dynamics simulations using NOE distance restraints for 2:1 palmatine-[d(TTGGGGT)]4 complex reveal end-stacking of palmatine at G6pT7 step and groove binding along T2pG3 step. Binding to [d(TTAGGGT)]4 takes place at T2pA3pG4 and G6pT7 steps. Structural features of molecular recognition of two different G-quadruplex DNA sequences by palmatine have relevance in rational drug development for anti-cancer therapy.

Abstract P5-05-04: The APOBEC3B transgenic mouse

Abstract Background: Massively parallel DNA sequencing efforts have facilitated the production of catalogs of the somatic mutations present in cancer genomes. Examining the sequence context of the mutations it is possible to identify patterns that recur frequently. These mutational patterns or “signatures” enable inference into the mutational processes responsible for the patterns. Investigators at the Sanger Institute in the UK have produced a compendium of 30 “Signatures of Mutational Processes in Human Cancer”. Two of these signatures have been attributed to the actions of APOBEC cytidine deaminases. Data from other investigators suggest that APOBEC3B (A3B) is more likely to play a role than are other members of the APOBEC family. The evidence to support the hypothesis that APOBEC activation is responsible for the mutation patterns is limited at present. It is important to establish whether these enzymes play a role in oncogenesis as the APOBEC mutational signature had been noted to be present in 1 of every 6 cancer specimens and second only to aging in prevalence. Methods: In order to determine if overexpression of APOBEC3B in the mammary gland of mice is tumorigenic and produces the mutational signatures attributed to its activity, we contracted with a commercial vendor for the production of an APOBEC3B transgenic mouse on a 129:C57BL/6 background. Of note, in the course of the production of the transgenic, the intron between exons 5 and 6 was retained in the construct to prevent the expression of the highly mutatgenic protein in E. coli. The transgene was placed 3' of a stop sequence flanked by LoxP sites within the ROSA26 locus. Mating with MMTV-Cre mice restricts the excision of the stop cassette and expression of APOBEC3B to the mammary tissue. A3B heterozygotes from the F1 generation were bred to produce A3B homozygotes. Homozygotes have been allowed to undergo multiple pregnancies and have been observed for up to 12 months as of June 2017. Results: 3 mice have developed malignancies. Two mice appear to have developed lymphomas and the epithelial cells of a mammary gland of a third mouse (homozygote) at 11 months of age are diffusely hyperplastic. The cells are arranged in rows ranging from 2-4 cells in thickness. Periodic acid–Schiff (PAS) staining confirms that the basement membrane is intact around the hyperplasic cells. There is no anisocytosis, anisokaryosis, desmoplasia, necrosis or mitotic figures noted in the gland. Conclusions: At 11 month of age, the mammary glands of APOBEC3B transgenic mice are beginning to display histologic abnormalities associated with breast oncogenesis. Observation will continue. With the passage of time we expect to observe more advanced and larger mammary lesions. DNA will be extracted from any invasive mammary tumors, sequenced and the mutation signature determined using the EMu: Expectation-Maximisation inference of mutational signatures software available from Sanger. Citation Format: Choi M, Lyle LT, Song C, Clare SE. The APOBEC3B transgenic mouse [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-04.