Gene Tagging Research Articles

Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

Leishmania parasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited in Leishmania because of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Beneke et al. and the glmS ribozyme-based knock-down system in the model parasite Leishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in alternative media and identified supplemented MEM, IMDM and McCoy’s 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentration-dependent down-regulation of the episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.

CRISPR-mediated knock-in in the mouse embryo using long single stranded DNA donors synthesised by biotinylated PCR.

Successful gene knock-in by CRISPR-Cas9 in the mouse zygote requires three components; guideRNA, Cas9 protein and a suitable donor template, which usually comprises homology flanked insert sequence. Recently, long single stranded DNA (lssDNA) donors have emerged as a popular choice of DNA donor, outperforming dsDNA templates in terms of knock-in efficiency for gene tagging and generating conditional alleles. The generation of these donors can be achieved through several methods that may introduce errors in the sequence, result in poor yields, and contain dsDNA contamination. We have developed our own cost-effective lssDNA synthesis methodology that results in high purity, sequence verified, low contamination lssDNA donors. We provide a detailed methodology on the design and generation of such donors for gene tagging experiments and generating conditional alleles.

The biogeochemical variability of Arctic thermokarst ponds is reflected by stochastic and niche-driven microbial community assembly processes.

SummaryShallow thermokarst ponds are a conspicuous landscape element of the Arctic Siberian tundra with high biogeochemical variability. Little is known about how microbes from the regional species pool assemble into local pond communities and how the resulting patterns affect functional properties such as dissolved organic carbon (DOC) remineralization and greenhouse gas (GHG) turnover. We analysed the pelagic microbiomes of 20 ponds in north‐eastern Siberia in the context of their physico‐chemical properties. Ponds were categorized as polygonal or trough according to their geomorphological origin. The diversity of bacteria and eukaryotic microbes was assessed by ribosomal gene tag sequencing. Null model analysis revealed an important role of stochastic assembly processes within ponds of identical origin, in particular for genotypes only occurring in few systems. Nevertheless, the two pond types clearly represented distinct niches for both the bacterial and eukaryotic microbial communities. Carbon dioxide concentration, indicative of heterotrophic microbial processes, varied greatly, especially in the trough ponds. Methane concentrations were lower in polygonal ponds and were correlated with the estimated abundance of methanotrophs. Thus, the overall functional variability of Arctic ponds reflects the stochastic assembly of their microbial communities. Distinct functional subcommunities can, nevertheless, be related to GHG concentrations.

Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Salmonella enterica Serovar Typhimurium

To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or λ red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on the Salmonella enterica serovar Typhimurium chromosome. The method combines an efficient seamless assembly system in vitro, red homologous recombination in vivo, and counterselection marker sacB. To test this method, with the seamless assembly system, various modification fragments for target genes cpxR, cpxA, and acrB were rapidly and efficiently constructed in vitro. sacBKan cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of Salmonella Typhimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace the sacBKan cassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 × FLAG tagging of the target genes. This method can be used in any bacterial species that supports sacB gene activity and λ red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

Ginsenosides in root exudates of Panax notoginseng drive the change of soil microbiota through carbon source different utilization

Ginsenosides are the main bioactive components of Panax plants which could be secreted by root and show autotoxicity to root cells or promote the growth of soil-borne pathogens. However, comprehensive understanding of the effect of ginsenosides on soil microbiota is still lacking. The ginsenosides in root exudates of P. notoginseng were quantified and exogenous ginsenosides on soil microbiota were tested using 16S rRNA and ITS gene tag sequencing. Then its underlying mechanism was deciphered through studying effects of ginsenosides on growth of the ginsenoside-modified culturable fungi and bacteria as well as the relationships between these fungi and bacteria. Exogenous root exudates and mixtures of Rg1 + Rb1 + Rd had similar ability to drive the change of soil microbiota. Further studies demonstrated that Rg1 + Rb1 + Rd mixture could enrich or suppress special fungi and bacteria to modify soil community through differential utilization of carbon source during the early stage (30 days), followed by antagonism between ginsenoside-modified fungi and bacteria to determine soil microbial community modification at later stage (60 and 90 days). Ginsenosides were the main substances in exogenous root exudates of P. notoginseng that drove the change in soil microbiota, mediating the special interaction between the plant and the microbiota.

ARF-AID: A Rapidly Inducible Protein Degradation System That Preserves Basal Endogenous Protein Levels.

Inducible degron systems are widely used to specifically and rapidly deplete proteins of interest in cell lines and organisms. An advantage of inducible degradation is that the biological system under study remains intact and functional until perturbation, a feature that necessitates that the endogenous levels of the protein are maintained. However, endogenous tagging of genes with auxin‐inducible degrons (AID) can result in chronic, auxin‐independent proteasome‐mediated degradation. The ARF‐AID (auxin‐response factor–auxin‐inducible degron) system is a re‐engineered auxin‐inducible protein degradation system. The additional expression of the ARF‐PB1 domain prevents chronic, auxin‐independent degradation of AID‐tagged proteins while preserving rapid auxin‐induced degradation of tagged proteins. Here, we describe the protocol for engineering human cell lines to implement the ARF‐AID system for specific and inducible protein degradation. These methods are adaptable and can be extended from cell lines to organisms. © 2020 The Authors. Basic Protocol 1: Generation of ARF‐P2A‐TIR1 progenitor cells Basic Protocol 2: Designing, cloning, and testing of a gene‐specific sgRNA Basic Protocol 3: Design and amplification of a homology‐directed repair construct (C‐terminal tagging) Alternate Protocol 1: Design and amplification of a homology‐directed repair construct (N‐terminal tagging) Basic Protocol 4: Tagging of a gene of interest with AID Alternate Protocol 2: Establishment of an ARF‐AID clamp system Basic Protocol 5: Testing of auxin‐mediated degradation of the AID‐tagged protein

An optimized QF-binary expression system for use in zebrafish

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.

Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes.

Fluorescence microscopy has become a powerful tool in molecular cell biology. Visualizing specific proteins in bacterial cells requires labeling with fluorescent or fluorogenic tags, preferentially at the native chromosomal locus to preserve expression dynamics associated with the genomic environment. Exploring protein function calls for targeted mutagenesis and observation of differential phenotypes. In the model bacterium Escherichia coli, protocols for tagging genes and performing targeted mutagenesis currently involve multiple steps. Here, we present an approach capable of simultaneous tagging and mutagenesis of essential and nonessential genes in a single step. We require only the insertion of a stretch of the target gene into an auxiliary plasmid together with the tag. Recombineering-based exchange with the native locus is then carried out, where the desired mutation is introduced during amplification with homology-bearing primers. Using this approach, multiple tagged mutants per gene can be derived quickly.

Bacterial expression of 183-227aa region of HER3 extracellular domain I and preparation and identification of its polyclonal antibodies

To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in E.coli BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.

Endogenous p21-Dependent Transgene Control for CHO Cell Engineering.

Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins. However, the dynamic regulation of transgene expression is limited in current systems. Here, we investigated the effective regulation of transgene expression in CHO cells via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2 into the p21 locus effectively reduced the apoptosis, compared with random populations in which Bcl-2 expression was driven by cytomegalovirus (CMV) promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the antiapoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. We further demonstrated the inducible transgene expression by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with cellular states.

Efficient targeted integration directed by short homology in zebrafish and mammalian cells.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

CRISPR-Cas12a-assisted PCR tagging of mammalian genes.

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.

Improvement of Rice Breeding for Salinity Tolerance

Rice is one of the most significant food crop of the world. The production of rice is the second-highest in the world. Salinity is a serious constraint to increase rice production worldwide. Review of recent efforts achieved towards rice breeding for salinity tolerance has a paramount importance beside for rice production improvement in Ethiopia. Rice is a relatively new crop to Ethiopia; however, the crop is relatively well adapted to different parts of the country. Salinity is a problem of many irrigated, arid and semi-arid areas of the world where rain fall is insufficient to leach out salts from the root zone. Typical symptoms of salt injury in rice are stunted growth, leaf rolling, white leaf tip, white blotches in the leaf blade, drying of the older leaves and poor root growth. The main mechanisms of salt tolerance are control of salt uptake, reducing damage under excessive ion uptake and osmotic adjustment. The three major breeding approaches for salinity tolerant varietal development are conventional breeding, marker assisted selection and genetic engineering. Conventional rice breeding approaches exercised for thousands of years. But, the progress is slow, due to the time consuming process, the quantitative nature of most agronomic traits make difficulties in genotypes selection. However, modern breeding is more precise and efficient and easily identify undesirable genes that can be eliminated in future generations. In rice, markers like RAPDs, AFLPs, ISSRs and SSRs have been used. Among these DNA markers, SSRs is highly preferable for gene tagging and mapping efforts. The development of salt-tolerant variety by transgenic way needs more investigation. To date, transgenic salt tolerant variety has not yet reached at farmers’ field. In Ethiopia a little beat was done to identify rice genotypes adaptable to adverse saline soil conditions. From here on, more research findings highly crucial for the development of salinity tolerant rice varieties in the country. Therefore, it is a good opportunity for rice breeders to develop salt tolerant high yielding varieties in Ethiopia. Keywords : Conventional breeding, Genetic engineering, Marker-assisted selection, Rice, Salt tolerance DOI: 10.7176/JEES/10-5-02 Publication date: May 31 st 2020

Vertical zonation of bacterial assemblages attributed to physical stratification during the summer relaxation of the coastal upwelling off Galicia (NW Spain)

We combined flow cytometry, CARD-FISH, and 16S rRNA gene tag pyrosequencing to investigate bacterioplankton dynamics along a transect in shelf waters off A Coruña (Galicia, NW Spain). Over five days (16-20th July 2012) we sampled during the relaxation of a summer upwelling pulse, providing an opportunity to examine the impact of pulses of cold nutrient-rich water into coastal microbial communities. The hydrographic conditions, characterized by intense density stratification of surface waters and the presence of a deep chlorophyll maximum (DCM) at 20–30 m, were relatively maintained over the sampling period. Indeed, bacterial abundance and composition displayed low day to day variation. Alpha diversity analysis suggested that species richness and diversity increased from coastal to shelf stations and from the surface down to the coastal DCM, which could be caused by the mixing of upwelled bacteria with the coastal surface waters. SAR11, SAR86, and Roseobacter were the most abundant bacteria detected in the samples by using CARD-FISH. The assemblages observed by pyrosequencing displayed a strong vertical zonation along the transect. Rhodobacteraceae (under class Alphaproteobacteria) and Bacteriodetes dominated the surface waters and decreased during the upwelling pulse, while SAR 86 (under class Gammaproteobacteria), Actinobacteria and SAR11 clade increased their relative abundance at the coastal DCM with upwelling relaxation, particularly at the shelf stations. Bacterial assemblages from surface waters were associated with higher temperature and light conditions, while coastal DCM assemblages were rather associated to salinity, inorganic nutrients and a diatom-bloom leading to high chlorophyll-a. Our findings suggest that the vertical variability in environmental conditions induced by the intense density stratification, the exportation of warmer and less saline surface water from the rias to the adjacent shelf, and the fertilizing effect of recently upwelled water at the deeper layer, determined the composition of distinct bacterial assemblages at the subsurface and DCM layers.

Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).

Lifelong Analysis of Key Aging Genes as Determinants of Lifespan in C. elegans

Aging is an integrative phenotype subject to a complex interplay of genetic, environmental, and life history factors, and a key risk factor for a multitude of human diseases. Research in model organisms has enabled the identification of key evolutionary conserved genetic pathways that play a role in aging. In particular, research on the model organism Caenorhabditis elegans has been crucial in our current understanding of the genetic and environmental regulation of lifespan. Although a multitude of pathways are known to affect longevity, how these pathways jointly respond to upstream stimuli, and how they integrate this information to drive lifespan is far from understood. A major limitation to answer this question is the technical difficulty associated with studying the spatiotemporal activity of multiple pathways throughout lifespan, and under a variety of environmental conditions. In this work, we present a system that enables in vivo tracking the endogenous spatiotemporal activity of key aging genes throughout C. elegans lifespan. This system hinges on an integrative experimental platform based on microfluidics, computer vision, and tagging of endogenous genes via CRISPR/Cas9 genetic engineering approaches. In contrast to traditional transgene expression, CRISPR/Cas9 enables insertion of a tag at precise genomic locations. This results in fluorescent protein levels representative of the endogenously expressed genes, and where all isoforms can be analyzed. Studying endogenous protein levels, however, poses a significant challenge, as these reporters are extremely dim in comparison to traditional multi‐copy insertion transgenes. To address this limitation, we have developed computer vision approaches to quantitatively determine the spatial location and levels of said proteins, which can be used as a metric for gene activity. Furthermore, the use of microfluidic devices enables culture, stimulation, and longitudinal high‐resolution imaging of animal populations under precise environmental conditions. Taking advantage of our computer vision algorithms, we can quantify protein levels, cellular compartmentalization, and tissue localization. Using this approach, we have studied the key transcription factor, DAF‐16/FOXO, the main regulator of Insulin/Insulin‐like Signaling in C. elegans. Under a variety of exposures to dietary restriction, a well‐known regulator of lifespan that acts through DAF‐16, we have observed patterns of activity that have not been identified with traditional transgenes. Integrating lifespan measurements under varied environmental conditions with quantitative analysis from DAF‐16 lifelong spatiotemporal activity, we are exploring the predictive power of this key transcription factor at the tissue‐level, using statistical and mathematical models. We are working on expanding our analysis to additional lifespan regulators to better understand how these interact in driving lifespan.Support or Funding InformationThis work was supported in part by grants NIH R21AG059099 and NSF 1838314

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.

Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

The Escherichia coli alkA Gene Is Activated to Alleviate Mutagenesis by an Oxidized Deoxynucleoside.

The cellular methyl donor S-adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (m3A), which in Escherichia coli is removed by m3A-DNA glycosylase I (Tag) and II (AlkA). The tag gene is constitutively expressed, while alkA is induced by sub-lethal concentrations of methylating agents. We previously found that AlkA exhibits activity for the reactive oxygen-induced thymine (T) lesion 5-formyluracil (fU) in vitro. Here, we provide evidence for AlkA involvement in the repair of oxidized bases by showing that the adenine (A) ⋅ T → guanine (G) ⋅ cytosine (C) mutation rate increased 10-fold in E. coli wild-type and alkA– cells exposed to 0.1 mM 5-formyl-2′-deoxyuridine (fdU) compared to a wild-type specific reduction of the mutation rate at 0.2 mM fdU, which correlated with alkA gene induction. G⋅C → A⋅T alleviation occurred without alkA induction (at 0.1 mM fdU), correlating with a much higher AlkA efficiency for fU opposite to G than for that to A. The common keto form of fU is the AlkA substrate. Mispairing with G by ionized fU is favored by its exclusion from the AlkA active site.

Development and Characterization of Simple Sequence Repeat Markers for, and Genetic Diversity Analysis of Liquidambar formosana

Liquidambar formosana (Hamamelidaceae) is a relatively fast-growing deciduous tree of high ornamental value that is indigenous to China. However, few molecular markers are available for the species or its close relatives; this has hindered genomic and genetic studies. Here, we develop a series of transferable expressed sequence tag-simple sequence repeats (EST-SSRs) for genomic analysis of L. formosana. We downloaded the sequence of the L. formosana transcriptome from the National Center of Biotechnology Information Database and identified SSR loci in the Unigene library. We found 3284 EST-SSRs by mining 34,491 assembled unigenes. We synthesized 100 random primer pairs for validation of eight L. formosana individuals; of the 100 pairs, 32 were polymorphic. We successfully transferred 12 EST-SSR markers across three related Liquidambar species; the markers exhibited excellent cross-species transferability and will facilitate genetic studies and breeding of Liquidambar. A total of 72 clones of three Liquidambar species were uniquely divided into three main clusters; principal coordinate analysis (PCoA) supported this division. Additionally, a set of 20 SSR markers that did not exhibit nonspecific amplification were used to genotype more than 53 L. formosana trees. The mean number of alleles (Na) was 5.75 and the average polymorphism information content (PIC) was 0.578, which was higher than that of the natural L. formosana population (0.390). In other words, the genetic diversity of the plus L. formosana population increased, but excellent phenotypic features were maintained. The primers will be valuable for genomic mapping, germplasm characterization, gene tagging, and further genetic studies. Analyses of genetic diversity in L. formosana will provide a basis for efficient application of genetic materials and rational management of L. formosana breeding programs.

Dynamics and ecological distributions of the Archaea microbiome from inland saline lakes (Monegros Desert, Spain).

We characterized the rich Archaea microbiome of shallow inland lakes (Monegros Desert, NE Spain) by 16S rRNA gene tag sequencing covering a wide salinity range (0.1%-40% w/v) along 3 years. Up to 990 operational taxonomic units (OTUs; >97% identity) were detected allocated in 14 major archaeal phyla and heterogeneously distributed along the salt gradient. Dynamics and idiosyncratic ecological distributions were uncovered for the different phyla. A high genetic richness was observed for Woesearchaeota and Pacearchaeota (>370 OTUs each), followed by Halobacteria (105), Nanohaloarchaeota (62) and Thermoplasmata (19). Overall, the distribution of genetic richness was strongly correlated with environmental niche amplitude, but not with occurrence. We unveiled high occurrence for a very rich Woesearchaeota assemblage, and an unexpected positive correlation of Pacearchaeota abundance with salinity at >15%dissolved salt content. The estimated dynamic behaviour (temporal 'turnover' rates of presence/absence data) unveiled Thaumarchaeota and Halobacteria as the most dynamic groups, and Aenigmarchaeota and Thermoplasmata as the most stable. The DPANN Pacearchaeota, Woesearchaeota, and Nanohaloarchaeota showed intermediate rates, suggesting higher resilience to environmental perturbations. A rich and dynamic Archaea microbiome was unveiled, including unseen ecological traits for relevant members of the still largely unknown DPANN group, supporting a strong ecological differentiation between Pacearchaeota and Woesearchaeota.