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Mutagenic purine–pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine–pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine–pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

SummaryMyometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, which are benign tumors that develop in the myometrium of most reproductive age women, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better discerning markers for more rigorous downstream analyses. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers capable of further enriching for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers, includingSUSD2. CRIP1expression was found highly upregulated in both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Early organismal development consists of transformative events that lay the foundation for body formation and long-term phenotype. Despite this understanding, the rapid progression of events and the limited material available are major barriers to studying the earliest stages. The size and accessibility of Drosophila embryos overcome some of these limitations, and several studies characterizing early transcriptional events have been reported. Unfortunately, manual embryo staging, and elaborate protocols make the techniques employed in these studies prone to human and technical errors and incompatible with routine laboratory use. Herein, we present a straight-forward and operationally simple methodology for studying the early transcription (≤3 hours) in Drosophila. This method relies on single-embryo RNA-sequencing and transcriptome ordering along a developmental trajectory (pseudo-time), thereby avoiding the need for the staging of the embryos. The obtained high-resolution and time-sensitive mRNA expression profiles uncovered the exact onset of transcription and degradation of transcripts and revealed an earlier transcription start for several zygotic genes. In addition, degradation patterns suggest that maternal mRNA decay is independent of mRNA levels. By classifying each embryo as male or female, we were also able to study sex-biased transcription between the beginning of zygotic transcription to gastrulation and identified 120 differentially expressed mRNAs. Using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y-chromosome genotyping. Herein, we report an accessible, single-embryo sequencing approach for high-resolution, time-sensitive transcriptome analysis. Our data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) – including β-hydroxybutyrate (βOHB) and acetoacetate (AcAc) – as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and βOHB directly increases CD8+ Teff cell IFN-γ production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that βOHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

SummaryAntibody-secreting cells (ASCs) are key contributors to humoral immunity through immunoglobulin production and the potential to be long-lived. ASCs have been recently identified in the thymus (THY) which is of interest as autoreactive THY B cells regulate T cell tolerance. Here, we showed that the young female THY was skewed towards higher production of ASCs relative to males. However, these differences disappeared with age. Intravenous labeling combined with blockade of CD154(CD40L) suggested that THY ASCs were generatedin situin a T cell dependent fashion. Single cell RNA-sequencing revealed that THY ASCs possessed a transcriptional signature indicative of interferon signaling and activation. Flow cytometry confirmed that THY ASCs had increased levels of Toll-like receptor 7 as well as the activation markers CD69 and major histocompatibility class II. Overall, THY ASCs are a unique population that require further investigation to fully appreciate their biological significance.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

Chiococca alba (L.) Hitchc. (snowberry), a member of the Rubiaceae, has been used as a folk remedy for a range of health issues including inflammation and rheumatism and produces a wealth of specialized metabolites including terpenes, alkaloids, and flavonoids. We generated a 558 Mb draft genome assembly for snowberry which encodes 28,707 high-confidence genes. Comparative analyses with other angiosperm genomes revealed enrichment in snowberry of lineage-specific genes involved in specialized metabolism. Synteny between snowberry and Coffea canephora Pierre ex A. Froehner (coffee) was evident, including the chromosomal region encoding caffeine biosynthesis in coffee, albeit syntelogs of N-methyltransferase were absent in snowberry. A total of 27 putative terpene synthase genes were identified, including 10 that encode diterpene synthases. Functional validation of a subset of putative terpene synthases revealed that combinations of diterpene synthases yielded access to products of both general and specialized metabolism. Specifically, we identified plausible intermediates in the biosynthesis of merilactone and ribenone, structurally unique antimicrobial diterpene natural products. Access to the C. alba genome will enable additional characterization of biosynthetic pathways responsible for health-promoting compounds in this medicinal species.

Switchgrass ( is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice ( L.), maize ( L.), soybean [ (L.) Merr.], and Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass.

Introduction: Hash functions are computer algorithms that protect information and secure transactions. In response to the NIST`s "International Call for Hash Function", we developed a biological hash function using the computing capabilities of bacteria. We designed a DNA-based XOR logic gate that allows bacterial colonies arranged in a series on an agar plate to perform hash function calculations. Results and Discussion: In order to provide each colony with adequate time to process inputs and perform XOR logic, we designed and successfully demonstrated a system for time-delayed bacterial growth. Our system is based on the diffusion of -lactamase, resulting in destruction of ampicillin. Our DNA-based XOR logic gate design is based on the op-position of two promoters. Our results showed that and functioned as expected individually, but did not behave as expected in the XOR construct. Our data showed that, contrary to literature reports, the promoter is bidirectional. In the absence of the 3OC6 inducer, the LuxR activator can bind to the promoter and induce backwards transcription. Conclusion and Prospects: Our system of time delayed bacterial growth allows for the successive processing of a bacterial hash function, and is expected to have utility in other synthetic biology applications. While testing our DNA-based XOR logic gate, we uncovered a novel function of . In the absence of autoinducer 3OC6, LuxR binds to and activates backwards transcription. This result advances basic research and has important implications for the widespread use of the promoter.

Sweet potato (Ipomoea batatas [L.] Lam.) is an important subsistence crop in Sub‐Saharan Africa, yet as for many crops, yield can be severely impacted by drought stress. Understanding the genetic mechanisms that control drought tolerance can facilitate the development of drought‐tolerant sweet potato cultivars. Here, we report an expression profiling study using the US‐bred cultivar, Beauregard, and a Ugandan landrace, Tanzania, treated with polyethylene glycol (PEG) to simulate drought and sampled at 24 and 48 hr after stress. At each time‐point, between 4,000 to 6,000 genes in leaf tissue were differentially expressed in each cultivar. Approximately half of these differentially expressed genes were common between the two cultivars and were enriched for Gene Ontology terms associated with drought response. Three hundred orthologs of drought tolerance genes reported in model species were identified in the Ipomoea trifida reference genome, of which 122 were differentially expressed under at least one experimental condition, constituting a list of drought tolerance candidate genes. A subset of genes was differentially regulated between Beauregard and Tanzania, representing genotype‐specific responses to drought stress. The data analyzed and reported here provide a resource for geneticists and breeders toward identifying and utilizing drought tolerance genes in sweet potato.

The Escherichia coli lac Operon is controlled by a regulatory system that has been the subject of intensive study for the past fifty years. The system creates metabolic efficiency by responding to the levels of environmental lactose. In the absence of lactose, the LacI protein acts as a repressor of transcription from the lac promoter. Transcription begins when lactose binds to LacI, which results in the expression of three genes involved in lactose uptake and catabolism. The lac promoter is the most commonly used promoter in the field of synthetic biology. Although it is widely used, the lac promoter is known to have leaky transcription, meaning that transcription takes place even when the repressor is present and the inducer is absent. In an effort to redesign the lac promoter, we tested pLac variants that were reported to have a higher affinity for RNA polymerase than the wild-type. We also compared three mutants of the LacI repressor that were reported to have increasing affinity for the pLa...

Synthetic biologists have adopted the engineering principle of standardization of parts and assembly in the construction of a variety of genetic circuits that program living cells to perform useful tasks. In this chapter, we describe the BioBrick standard as a widely used method. We present methods by which new BioBrick parts can be designed and produced, starting with existing clones, naturally occurring DNA, or de novo. We detail the procedures by which BioBrick parts can be assembled into construction intermediates and into biological devices. These protocols are based on our experience in conducting synthetic biology research with undergraduate students in the context of the iGEM competition.