Species-specific KRAB-ZFPs function as repressors of retroviruses by targeting PBS regions.

SignificanceHosts often target the relatively conserved regions in rapidly mutating retroviruses to inhibit their replication. One of these regions is called a primer binding site (PBS), which has to be complementary to the host tRNA to initiate reverse transcription. By analyzing endogenous retroviral elements, we found that host cells use this sequence as a target in efforts to block the expression of viral elements. A specific type of zinc finger protein targets the PBS in a host genome, which not only inhibits the transcription of endogenous viruses but also inhibits the replication of exogenous retroviruses with the same PBS. Thus, our study sheds light on a strategy for searching for host restriction factors targeting retroviruses.

A novel adenylate isopentenyltransferase 5 regulates shoot branching via the ATTTA motif in Camellia sinensis.

BACKGROUND: Shoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated. RESULTS: In this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3' untranslated region (3' UTR) and 5' UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3' UTR AS variant 2 (3AS2) might act as the predominant AS transcript. CONCLUSIONS: Together, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.

DAP-Seq Identification of Transcription Factor-Binding Sites in Potato.

Plant growth and adaptation to environmental fluctuations involve a tight control of cellular processes which, to a great extent, are mediated by changes at the transcriptional level. This regulation is exerted by transcription factors (TFs), a group of regulatory proteins that control gene expression by directly binding to the gene promoter regions via their cognate TF-binding sites (TFBS). The nature of TFBS defines the pattern of expression of the various plant loci, the precise combinatorial assembly of these elements being key in conferring plant's adaptation ability and in domestication. As such, TFs are main potential targets for biotechnological interventions, prompting in the last decade notable protein-DNA interaction efforts toward definition of their TFBS. Distinct methods based on in vivo or in vitro approaches defined the TFBS for many TFs, mainly in Arabidopsis, but comprehensive information on the transcriptional networks for many regulators is still lacking, especially in crops. In this chapter, detailed protocols for DAP-seq studies to unbiased identification of TFBS in potato are provided. This methodology relies on the affinity purification of genomic DNA-protein complexes in vitro, and high-throughput sequencing of the eluted DNA fragments. DAP-seq outperforms other in vitro DNA-motif definition strategies, such as protein-binding microarrays and SELEX-seq, since the protein of interest is directly bound to the genomic DNA extracted from plants yielding all the potential sites bound by the TF in the genome. Actually, data generated from DAP-seq experiments are highly similar to those out of ChIP-seq methods, but are generated much faster. We also provide a standard procedure to the analysis of the DAP-seq data, addressed to non-experienced users, that involves two consecutive steps: (1) processing of raw data (trimming, filtering, and read alignment) and (2) peak calling and identification of enriched motifs. This method allows identification of the binding profiles of dozens of TFs in crops, in a timely manner.

OPT gene family analysis of potato (Solanum tuberosum) responding to heavy metal stress: Comparative omics and co-expression networks revealed the underlying core templates and specific response patterns.

Oligopeptides transporter (OPT) can maintain intracellular metal homeostat, however, their evolutionary characteristics, as well as their expression patterns in heavy metal exposure, remain unclear. Compared with previous OPT family identification, we identified 94 OPT genes (including 21 in potato) in potato and 4 other plants by HMMER program based on OPT domain (PF03169) for the first time. Secondly, conserved and special OPTs were found through comprehensive analysis. Thirdly, spatio-temporal tissue specific expression patterns and co-expression frameworks of potato OPT genes under different heavy metal stress were constructed. These data can provide excellent gene resources for food security and soil remediation.

Molecular characterization of the COPT/Ctr-type copper transporter family under heavy metal stress in alfalfa.

In nature, heavy metals significantly affect crop growth and quality. Among various heavy metals, copper (Cu) is both essential and toxic to plants depending on the concentration and complex homeostatic networks. The Cu transporter family (COPT) plays important roles in Cu homeostasis, including absorption, transportation, and growth in plants; however, this gene family is still poorly understood in alfalfa (Medicago sativa L.). In this study, a total of 12 MsCOPTs were identified and characterized. Based on the conserved motif and phylogenetic analysis, MsCOPTs could be divided into four subgroups (A1, A2, A3, and B). Gene structure, chromosomal location, and synteny analyses of MsCOPTs showed that segmental and tandem duplications likely contributed to their evolution. Tissue-specific expression analysis of MsCOPT genes indicated diverse spatiotemporal expression patterns. Most MsCOPT genes had high transcription levels in roots and nodules, indicating that these genes may play vital roles in the absorption and transport of Cu through root. The complementary heterologous expression function of yeast once again indicates that root-specific COPT can supplement the growth of defective yeast strains on YPEG medium, suggesting that these genes are Cu transporters. In summary, for the first time, our research identified COPT family genes at the whole-genome level to provide guidance for effectively improving the problem of Cu deficiency in the grass-livestock chain and provide theoretical support for the subsequent development of grass and animal husbandry.

The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

YY1-mediated up-regulation of lncRNA LINC00466 facilitates glioma progression via miR-508/CHEK1.

BACKGROUND: The abnormal expression of lncRNA LINC00466 (LINC00466) has been demonstrated in several tumor types. However, the expression pattern and functions of LINC00466 in glioma remain uninvestigated. METHODS: A reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to analyze LINC00466 in human glioma tissues and cell lines. Luciferase reporter assays were performed to explore whether YY1 could bind to the promoter region of LINC00466. Cell counting kit-8, flow cytometry, colony-formation, transwell migration and invasion assays were carried out to determine the involvement of INC00466 in glioma. Luciferase assays and pulldown assays were conducted to verify the binding sites. RESULTS: We report that LINC00466 expression is increased in glioma cells and tissues. YY1 transcription factor (YY1) can bind directly to the LINC00466 promoter region. Clinical studies revealed that the elevated expression of LINC00466 is closely correlated with an advanced World Health Organization grade (p = 0.008), Karnofsky Performance Status score (p = 0.004) and a short overall survival (p = 0.0035) of glioma patients. Functional assays revealed that LINC00466 knockdown distinctly suppresses glioma cell proliferation, migration, invasion and epithelial-mesenchymal progress, and also promotes apoptosis. Moreover, dual-luciferase reporter assays indicated that LINC00466 acts as an endogenous sponge via binding to miR-508 and decreasing its expression. Luciferase assays and RT-PCR assays demonstrated that checkpoint kinase 1 (CHEK1) is a target of miR-508, and LINC00466 modulates CHEK1 levels by competing for miR-508. LINC00466 may exhibit its anti-oncogenic roles through targeting the miR-508/CHEK1 axis. CONCLUSIONS: Our findings identified a novel glioma-related long non-coding RNA, LINC00466, which may provide a potential novel prognostic and therapeutic target for glioma.

Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

Evolutionary history of the heat shock protein 90 (Hsp90) family of 43 plants and characterization of Hsp90s in Solanum tuberosum.

Heat shock protein 90 genes/proteins (Hsp90s) are related to the stress resistance found in various plant species. These proteins affect the growth and development of plants and have important effects on the plants under various stresses (cold, drought and salt) in the environment. In this study, we identified 334 Hsp90s from 43 plant species, and Hsp90s were found in all species. Phylogenetic tree and conserved domain database analysis of all Hsp90s showed three independent clades. The analysis of motifs, gene duplication events, and the expression data from PGSC website revealed the gene structures, evolution relationships, and expression patterns of the Hsp90s. In addition, analysis of the transcript levels of the 7 Hsp90s in potato (Solanum tuberosum) under low temperature and high temperature stresses showed that these genes were related to the temperature stresses. Especially StHsp90.2 and StHsp90.4, under high or low temperature conditions, the expression levels in leaves, stems, or roots were significantly up-regulated. Our findings revealed the evolution of the Hsp90s, which had guiding significance for further researching the precise functions of the Hsp90s.

OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways.

Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri).

Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11, PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological analysis confirmed that reduced PbrPME activities repressed the pollen tube growth. Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which deepened the understanding of pear fertility regulation.

Generation of fruit postharvest gene datasets and a novel motif analysis tool for functional studies: uncovering links between peach fruit heat treatment and cold storage responses.

MAIN CONCLUSION: A survey of developed fruit gene-specific datasets and the implementation of a novel cis-element analysis tool indicate specific transcription factors as novel regulatory actors under HT response and CI protection. Heat treatment (HT) prior to cold storage (CS) has been successfully applied to ameliorate fruit chilling injury (CI) disorders. Molecular studies have identified several HT-driven benefits and putative CI-protective molecules and mechanisms. However, bioinformatic tools and analyses able to integrate fruit-specific information are necessary to begin functional studies and breeding projects. In this work, a HT-responsive gene dataset (HTds) and four fruit expression datasets (FEds), containing gene-specific information from several species and postharvest conditions, were developed and characterized. FEds provided information about HT-responsive genes, not only validating their sensitivity to HT in different systems but also revealing most of them as CS-responsive. A special focus was given to peach heat treatment-sensitive transcriptional regulation by the development of a novel Perl motif analysis software (cisAnalyzer) and a curated plant cis-elements dataset (PASPds). cisAnalyzer is able to assess sequence motifs presence, localization, enrichment and discovery on biological sequences. Its implementation for the enrichment analysis of PASPds motifs on the promoters of HTds genes rendered particular cis-elements that indicate certain transcription factor (TF) families as responsible of fruit HT-sensitive transcription regulation. Phylogenetic and postharvest expression data of these TFs showed a functional diversity of TF families, with members able to fulfil roles under HT, CS and/or both treatments. All integrated datasets and cisAnalyzer tool were deposited in FruitGeneDB (https://www.cefobi-conicet.gov.ar/FruitGeneDB/search1.php), a new available database with a great potential for fruit gene functional studies, including the markers of HT and CS responses whose study will contribute to unravel HT-driven CI-protection and select tolerant cultivars.

Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress.

Potato crop requires high dose of nitrogen (N) to produce high tuber yield. Excessive application of N causes environmental pollution and increases cost of production. Hence, knowledge about genes and regulatory elements is essential to strengthen research on N metabolism in this crop. In this study, we analysed transcriptomes (RNA-seq) in potato tissues (shoot, root and stolon) collected from plants grown in aeroponic culture under controlled conditions with varied N supplies i.e. low N (0.2 milli molar N) and high N (4 milli molar N). High quality data ranging between 3.25 to 4.93 Gb per sample were generated using Illumina NextSeq500 that resulted in 83.60-86.50% mapping of the reads to the reference potato genome. Differentially expressed genes (DEGs) were observed in the tissues based on statistically significance (p ≤ 0.05) and up-regulation with ≥ 2 log2 fold change (FC) and down-regulation with ≤ -2 log2 FC values. In shoots, of total 19730 DEGs, 761 up-regulated and 280 down-regulated significant DEGs were identified. Of total 20736 DEGs in roots, 572 (up-regulated) and 292 (down-regulated) were significant DEGs. In stolons, of total 21494 DEG, 688 and 230 DEGs were significantly up-regulated and down-regulated, respectively. Venn diagram analysis showed tissue specific and common genes. The DEGs were functionally assigned with the GO terms, in which molecular function domain was predominant in all the tissues. Further, DEGs were classified into 24 KEGG pathways, in which 5385, 5572 and 5594 DEGs were annotated in shoots, roots and stolons, respectively. The RT-qPCR analysis validated gene expression of RNA-seq data for selected genes. We identified a few potential DEGs responsive to N deficiency in potato such as glutaredoxin, Myb-like DNA-binding protein, WRKY transcription factor 16 and FLOWERING LOCUS T in shoots; high-affinity nitrate transporter, protein phosphatase-2c, glutaredoxin family protein, malate synthase, CLE7, 2-oxoglutarate-dependent dioxygenase and transcription factor in roots; and glucose-6-phosphate/phosphate translocator 2, BTB/POZ domain-containing protein, F-box family protein and aquaporin TIP1;3 in stolons, and many genes of unknown function. Our study highlights that these potential genes play very crucial roles in N stress tolerance, which could be useful in augmenting research on N metabolism in potato.

The transcription factor GATA10 regulates fertility conversion of a two-line hybrid tms5 mutant rice via the modulation of UbL40 expression.

The thermosensitive genic male sterile 5 (tms5) mutation causes thermosensitive genic male sterility in rice (Oryza sativa) through loss of RNase ZS1 function, which influences ubiquitin fusion ribosomal protein L40 (UbL40 ) messenger RNA levels during male development. Here, we used ATAC-seq, combined with analysis of H3K9ac and H3K4me2, to identify changes in accessible chromatin during fertility conversion of the two-line hybrid rice Wuxiang S (WXS) derived from a mutant tms5 allele. Furthermore, RNA-seq and bioinformatic analyses identified specific transcription factors (TFs) in differentially accessible chromatin regions. Among these TFs, only GATA10 targeted UbL40 . Osgata10 knockout mutations, which resulted in low expression of UbL40 and a tendency toward male fertility, confirmed that GATA10 regulated fertility conversion via the modulation of UbL40 . Meanwhile, GATA10 acted as a mediator for interactions with ERF65, which revealed that transcriptional regulation is a complex process involving multiple complexes of TFs, namely TF modules. It appears that the ERF141/MADS7/MADS50/MYB modules affect metabolic processes that control anther and pollen development, especially cell wall formation. Our analysis revealed that these modules directly or indirectly affect metabolic pathway-related genes to coordinate plant growth with proper anther development, and furthermore, that GATA10 regulates fertility conversion via the modulation of UbL40 expression.

Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5 ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3 In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.

A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177-182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure.

The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

Genome-wide identification, phylogeny, evolutionary expansion and expression analyses of bZIP transcription factor family in tartaty buckwheat.

BACKGROUND: In reported plants, the bZIP family is one of the largest transcription factor families. bZIP genes play roles in the light signal, seed maturation, flower development, cell elongation, seed accumulation protein, abiotic and biological stress and other biological processes. While, no detailed identification and genome-wide analysis of bZIP family genes in Fagopyum talaricum (tartary buckwheat) has previously been published. The recently reported genome sequence of tartary buckwheat provides theoretical basis for us to study and discuss the characteristics and expression of bZIP genes in tartary buckwheat based on the whole genome. RESULTS: In this study, 96 FtbZIP genes named from FtbZIP1 to FtbZIP96 were identified and divided into 11 subfamilies according to their genetic relationship with 70 bZIPs of A. thaliana. FtbZIP genes are not evenly distributed on the chromosomes, and we found tandem and segmental duplication events of FtbZIP genes on 8 tartary buckwheat chromosomes. According to the results of gene and motif composition, FtbZIP located in the same group contained analogous intron/exon organizations and motif composition. By qRT-PCR, we quantified the expression of FtbZIP members in stem, root, leaf, fruit, and flower and during fruit development. Exogenous ABA treatment increased the weight of tartary buckwheat fruit and changed the expressions of FtbZIP genes in group A. CONCLUSIONS: Through our study, we identified 96 FtbZIP genes in tartary buckwheat and synthetically further analyzed the structure composition, evolution analysis and expression pattern of FtbZIP proteins. The expression pattern indicates that FtbZIP is important in the course of plant growth and development of tartary buckwheat. Through comprehensively analyzing fruit weight and FtbZIP genes expression after ABA treatment and endogenous ABA content of tartary buckwheat fruit, ABA may regulate downstream gene expression by regulating the expression of FtPinG0003523300.01 and FtPinG0003196200.01, thus indirectly affecting the fruit development of tartary buckwheat. This will help us to further study the function of FtbZIP genes in the tartary buckwheat growth and improve the fruit of tartary buckwheat.

Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature.

Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5'-CGCTTTGCGACGCTCCCCTCCCTCCC-3') on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety.