Promoter Elements Research Articles

Identification and Bioinformatics Analysis on Phosphatidylcholine Diglyceride Choline Phosphotransferase Family Genes in Plants

Phosphatidylcholine diglyceride choline phosphotransferase (PDCT) is involved in the synthesis of seed triglyceride (TAG) in plant, catalyzing the transfer between phosphatidylcholine (PC) and diacylglycerol (DAG), and the loss of the PDCT activity reduces significantly the accumulation of seed polyunsaturated fatty acids (PUFAs). In this study, taking advantage of public available genomes of six oil crops (peanut, rape, soybean, cotton, sesame and sunflower), two staple food crops (rice and maize) and one aquatic plant (Chinese lotus), we identified in total 20 PDCT orthologues using bioinformatics, and further investigated into physical and protein chemical property, chromosome distribution, phylogenetic evolution, gene structure and promoter sequence. Our results showed that the PDCT  family members have conserved domains and gene structures, as well as stable protein properties and structures, implying certain evolutionary conservation among them. In addition, copy numbers of PDCT  were significantly different in tested plant species, while crop plants and Arabidopsis had one single copy, oil crops almost have at least 2 copies. Furthermore, the existence of many transcription core promoter elements and photo-response cis-acting elements in the promoter regions of these identified PDCT  genes indicated that the expression of PDCT  genes is likely affected by multiple developmental and environmental stress signals. In sum, this study provided a bioinformatics reference for further study on the potential application of PDCT  family genes in crop breeding.

ORGANIZATION OF THE 5S RIBOSOMAL DNA INTERGENIC SPACER OF APAMEA MONOGLYPHA (LEPIDOPTERA)

A significant part of the moth species, representatives of the superfamily Noctuoidea distributed on the territory of Ukraine, are pests of crops, which attracts special attention of researchers. In particular, host plants for the larvae of the Apamea species are cereals. For the correct identification of the pest moth speciesit is necessary to conduct molecular genetic studies to create molecular passports (barcoding). One of the convenient molecular markers used to compare low-ranking taxa is the 5S rDNA region represented by tandemly arranged clusters, where each repeat unit consists of a highly conserved coding region and a variable intergenic spacer (IGS). Since the molecular organization of 5S rDNA in moth species is still insufficiently studied, the aim of our work was to analyze the 5S rDNA IGS of A. monoglypha, a representative of the Apamea genus. For this, 5S rDNA repeats were amplified by PCR, and the obtained products were ligated into the pJet1.2 plasmid vector. The inserts of the three recombinant plasmids, ApMon1-1, ApMon1-2, and ApMon1-8, which were of the expected size, were sequenced. It was found that the A. monoglypha genome contains several structural IGS variants that differ both in length and nucleotide sequence. The level of similarity of the compared IGS sequences varied from 38.0 to 76.2%, indicating a high level of intragenomic polymorphism in this region. An AT-rich motif, a GC dinucleotide, and a cytosine residue up-stream of the coding region were identified as putative external promoter elements, which can be involved in the initiation of transcription. Furthermore, the oligo-T motif located immediately down-stream of the coding region is probably involved in the transcription termination.

Charged Amino Acids Contribute to ZorO Toxicity.

Chromosomally encoded toxin-antitoxin systems have been increasingly identified and characterized across bacterial species over the past two decades. Overproduction of the toxin gene results in cell growth stasis or death for the producing cell, but co-expression of its antitoxin can repress the toxic effects. For the subcategory of type I toxin-antitoxin systems, many of the described toxin genes encode a small, hydrophobic protein with several charged residues distributed across the sequence of the toxic protein. Though these charged residues are hypothesized to be critical for the toxic effects of the protein, they have not been studied broadly across different type I toxins. Herein, we mutated codons encoding charged residues in the type I toxin zorO, from the zor-orz toxin-antitoxin system, to determine their impacts on growth inhibition, membrane depolarization, ATP depletion, and the localization of this small protein. The non-toxic variants of ZorO accumulated both in the membrane and cytoplasm, indicating that membrane localization alone is not sufficient for its toxicity. While mutation of a charged residue could result in altered toxicity, this was dependent not only on the position of the amino acid within the protein but also on the residue to which it was converted, suggesting a complex role of charged residues in ZorO-mediated toxicity. A previous study indicated that additional copies of the zor-orz system improved growth in aminoglycosides: within, we note that this improved growth is independent of ZorO toxicity. By increasing the copy number of the zorO gene fused with a FLAG-tag, we were able to detect the protein expressed from its native promoter elements: an important step for future studies of toxin expression and function.

Knowledge, Attitude and Practice toward Nutrition among Communities in Southern Senatorial Zone of Borno State

Nutrition is a key element of health promotion, prevention, and management of diseases. The study was aimed at assessing the nutritional knowledge, attitude, and practice of communities and to determine factors influencing the nutritional knowledge, attitudes, and practices of these communities. A cross-sectional study was conducted among 10 communities. Multistage sampling was used to select the sample size of 1000 individuals. The study utilized both primary and secondary data through journals and administration of a self-developed structured interviewer questionnaire and focus group discussions. The data was analyzed using statistical package for social science (SPSS v20). Results were presented using descriptive statistics and Chi-square. The findings of the study revealed that communities have little or no knowledge about nutrition despite the availability of different sources of nutritional information, majority of communities have a negative attitude towards the importance of nutrition because of their Cultural beliefs. Moreso, majority communities do not prepare balanced family meals. The result from chi-square analysis shows that age and source of nutritional knowledge are the main factors influencing nutritional knowledge and education level, marital status, and sources of family income are the main factors influencing nutritional practices among communities. Base on the findings of the study it was recommended among others that there is need to increased public awareness and enlightenment to improve the attitude and practice of communities, community leaders and health workers to promote good dietary habits and consumption of good indigenous food to motivate practice among communities. Keywords: Attitude, Communities, Knowledge, Nutrition, Practice.

Novel perspectives on antisense transcription in HIV-1, HTLV-1, and HTLV-2.

The genome of retroviruses contains two promoter elements (called long terminal repeat or LTR) at the 5' and 3' end of their genome. Although the expression of retroviral genes generally depends on the promoter located in the 5' LTR, the 3' LTR also has promoter activity responsible for producing antisense transcripts. These natural antisense transcripts (NATs) are a class of RNA molecules transcribed from the opposite strand of a protein-coding gene. NATs have been identified in many prokaryotic and eukaryotic systems, as well as in human retroviruses such as human immunodeficiency virus type 1 (HIV-1) and HTLV-1/2 (human T-cell leukemia virus type 1/2). The antisense transcripts of HIV-1, HTLV-1, and HTLV-2 have been briefly characterized over the past several years. However, a complete appreciation of the role these transcripts play in the virus lifecycle and the cellular factors which regulate their transcription is still lacking. This review provides an overview of antisense transcription in human retroviruses with a specific focus on the MEF-2 family of transcription factors, the function(s) of the antisense protein products, and the application of antisense transcription models in therapeutics against HIV-1 and HTLV-1 in the context of co-infection.

SOX9 and HMGB3 co-operatively transactivate NANOG and promote prostate cancer progression.

The homeodomain-containing transcription factor NANOG is overexpressed in prostate adenocarcinoma (PCa) and predicts poor prognosis. The SOX family transcription factor SOX9, as well as the transcription co-activator HMGB3 of the HMGB family, are also overexpressed and may play pivotal roles in PCa. However, it is unknown whether SOX9 and HMGB3 interact with each other, or if they regulate NANOG gene transcription. We identified potential SOX9 responsive elements in NANOG promoter, and investigated if SOX9 regulated NANOG transcription in co-operation with HMGB3 by experimental analysis of potential SOX9 binding sites in NANOG promoter, reporter gene transcription assays with or without interference or artificial overexpression of SOX9 and/or HMGB3, and protein-binding assays of SOX9-HMGB3 interaction. Clinicopathologic and prognostic significance of SOX9-HMGB3 overexpression in PCa was analyzed. SOX9 activated NANOG gene transcription by preferentially binding to a highly conserved consensus cis-regulatory element (-573 to -568) in NANOG promoter, and promoted the expression of NANOG downstream oncogenic genes. Importantly, HMGB3 functioned as a partner of SOX9 to co-operatively enhance transactivation of NANOG by interacting with SOX9, predominantly via the HMG Box A domain of HMGB3. Overexpression of SOX9 and/or HMGB3 enhanced PCa cell survival and cell migration and were significantly associated with PCa progression. Notably, Cox proportional regression analysis showed that co-overexpression of both SOX9 and HMGB3 was an independent unfavorable prognosticator for both CRPC-free survival (relative risk [RR] = 3.779，95% confidence interval [CI]: 1.159-12.322, p = 0.028) and overall survival (RR = 3.615，95% CI: 1.101-11.876, p = 0.034). These findings showed a novel SOX9/HMGB3/NANOG regulatory mechanism, deregulation of which played important roles in PCa progression.

A promoter toolbox for tissue-specific expression supporting translational research in cassava (Manihot esculenta).

There is an urgent need to stimulate agricultural output in many tropical and subtropical countries of the world to combat hunger and malnutrition. The starchy crop cassava (Manihot esculenta), growing even under sub-optimal conditions, is a key staple food in these regions, providing millions of people with food. Cassava biotechnology is an important technique benefiting agricultural progress, but successful implementation of many biotechnological concepts depends on the availability of the right spatiotemporal expression tools. Yet, well-characterized cassava promoters are scarce in the public domain. In this study, we investigate the promoter activity and tissue specificity of 24 different promoter elements in stably transformed cassava plants. We show that many of the investigated promoters, especially from other species, have surprisingly low activity and/or tissue specificity, but feature several promoter sequences that can drive tissue-specific expression in either autotrophic-, transport- or storage tissues. We especially highlight pAtCAB1, pMePsbR, and pSlRBCS2 as strong and specific source promoters, pAtSUC2, pMeSWEET1-like, and pMeSUS1 as valuable tools for phloem and phloem parenchyma expression, and pStB33, pMeGPT, pStGBSS1, as well as pStPatatin Class I, as strong and specific promoters for heterotrophic storage tissues. We hope that the provided information and sequences prove valuable to the cassava community by contributing to the successful implementation of biotechnological concepts aimed at the improvement of cassava nutritional value and productivity.

HDAC19 recruits ERF4 to the MYB5a promoter and diminishes anthocyanin accumulation during grape ripening.

DNA acetylation alters the expression of responsive genes during plant development. In grapes (Vitis vinifera), however, little is known about this regulatory mechanism. In the present study, 'Kyoho' grapes treated with trichostatin A (TSA, a deacetylase inhibitor) were used for transcriptome sequencing and quantitative proteomics analysis. We observed that acetylation was associated with anthocyanin accumulation and gene expression. Acetylation positively regulated phenylalanine metabolism and flavonoid biosynthesis pathways. Using omics analysis, we detected an increase in the levels of the AP2/EREBP transcription factor family after TSA treatment, indicating its association with acetylation-deacetylation dynamics in grapes. Furthermore, ethylene response factor 4 (ERF4) physically interacted with VvHDAC19, a histone deacetylase, which synergistically reduced the expression of target genes involved in anthocyanin biosynthesis owing to the binding of VvERF4 to the GCC-box cis-regulatory element in the VvMYB5a promoter. VvHDAC19 and VvERF4 also controlled anthocyanin biosynthesis and accumulation by regulating acetylation levels of histones H3 and H4. Therefore, alterations in histone modification can significantly regulate the expression of genes involved in anthocyanin biosynthesis and affect grape ripening.

RpoN is required for the motility and contributes to the killing ability of Plesiomonas shigelloides

BackgroundRpoN, also known as σ54, first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides.ResultsTo identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT.ConclusionsWe showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes.

Paradigm and Framework of WUS-CLV Feedback Loop in Stem Cell Niche for SAM Maintenance and Cell Identity Transition

Shoot apical meristem (SAM) consists of stem cells that act as a reservoir for the aerial growth. It plays an important role in the differential architectural development in plants. SAM actively performs parallel functions by maintaining the pluripotent of stem cells and continuous organogenesis throughout the plant’s life cycle. Molecular mechanisms regulating the signaling networks of this dual function of the SAM have been progressively understood. In the SAM, the feedback loop of WUSCHEL (WUS)-CLAVATA (CLV) has been found to be the key regulator in stabilizing stem cell proliferation and differentiation. In general, WUS migrates into central zone (CZ) from organizing center (OC) and activates the expression of CLV3 by binding to the promoter elements. CLV3 acts as a ligand to interact with the CLV1, leucine rich repeats (LRR) receptor-like kinase (RLK) and LRR receptor-like protein CLV2, and protein kinase coryne (CRN) (CLV2/CRN) to restrict WUS transcription to the OC. Evolution of CLV3 is one of the main factors contributing to the transformation of two-dimensional (2D) to 3D plants. WUS-CLV loop is involved in several pathways and networks that integrate on meristem maintenance and cell identity transition. WUS-CLV maintains stem cells with simultaneous differentiation signals by the spatial-temporal signaling of the phytohormones. WUS-CLV loop has an interaction with reactive oxygen species (ROS), an important signaling molecules regulating cell proliferation and developmental transition. WUS also forms feedback loop with AGAMOUS (AG) for differentiation, proliferation, and termination of floral meristem. These loops might also involve in interaction with vernalization and its regulatory factors that oversees the precise timing of flowering after exposure to cold temperatures. In this review, we highlight the evolutionary and developmental importance of the WUS-CLV feedback loop on SAM maintenance and cell identity transition for inflorescence and floral meristem development.

Features of the regulation of the transcription factor NIN, which determined its participation in the control of nodule organogenesis in legumes plants

The transcription factor NIN (NODULE INCEPTION) is a master regulator of forming legume-rhizobial symbioses in plants. This transcriptional factor is the founder of the family of NIN-like proteins, which are widely represented in most part of groups of terrestrial plants, but only in legumes and some representatives of the Rosales was recruited to play a key role in the regulation of the development of rhizobial symbiosis. We assume that there are some features in the structure of the protein itself, which are associated with different co-regulators during various processes, which determines the selective involvement of NIN in the regulation of infection and organogenesis, and also there are many regulatory regions in the gene promoter that are associated with different TFs on different developmental stages of symbiosis.
 We found that this protein may contain structural features in the form of a specific transcription factor amino acid pattern that was characteristic of legumes with an indeterminate nodule type. In addition, we conducted a search and analysis of promoter elements, which showed activity in the roots of non-legume plants.
 In order to study the role of the identified promoter elements associated with cytokinins in the regulation of morphogenesis, we conducted an experiment on the treatment of mutant pea plants with exogenous cytokinins followed by transcriptomic analysis. As a result of this work, we have identified new target genes that can be activated by NIN in the control of later stages of nodule morphogenesis.
 The work was supported by the RSF grant 22-26-00279.

VvMYB14 participates in melatonin-induced proanthocyanidin biosynthesis by upregulating expression of VvMYBPA1 and VvMYBPA2 in grape seeds.

This work demonstrated that melatonin increases continuously in seeds, particularly seed coats, during berry ripening. Exogenous melatonin treatments significantly increased the proanthocyanidin (PA) content, partially through ethylene signaling, in seed coats. VvMYB14 expression exhibited patterns similar to melatonin accumulation over time, which was largely induced by melatonin treatment in seed coats during berry ripening. Additionally, VvMYB14 bound to the MBS element of the VvMYBPA1 promoter to activate expression. VvMYB14 overexpression largely upregulated expression of VvMYBPA1, VvMYBPA2 and VvLAR1 and increased the PA content in grape seed-derived calli. Similar increases in AtTT2 and AtBAN expression and PA content were found in VvMYB14-overexpressing Arabidopsis seeds. It was also observed that VvMYB14 overexpression increased ethylene production and thereby induced expression of VvERF104, which bound to the ERF element of the VvMYBPA2 promoter and activated its expression. Additionally, VvERF104 suppression reduced the VvMYB14 overexpression-induced increases in expression of VvMYBPA2 and VvLAR1 and PA content. Further experiments revealed that melatonin-induced increases in the expression of VvMYBPA1, VvMYBPA2, VvERF104 and VvLAR1 and PA accumulation were significantly reduced in VvMYB14-suppressing grape calli and leaves. Collectively, VvMYB14 mediates melatonin-induced PA biosynthesis by directly transactivating VvMYBPA1 expression and indirectly upregulating VvMYBPA2 expression via VvERF104.

A superior gene allele involved in abscisic acid signaling enhances drought tolerance and yield in chickpea.

Identifying potential molecular tags for drought tolerance is essential for achieving higher crop productivity under drought stress. We employed an integrated genomics-assisted breeding and functional genomics strategy involving association mapping, fine mapping, map-based cloning, molecular haplotyping and transcript profiling in the introgression lines (ILs)- and near isogenic lines (NILs)-based association panel and mapping population of chickpea (Cicer arietinum). This combinatorial approach delineated a bHLH (basic helix-loop-helix) transcription factor, CabHLH10 (Cicer arietinum bHLH10) underlying a major QTL, along with its derived natural alleles/haplotypes governing yield traits under drought stress in chickpea. CabHLH10 binds to a cis-regulatory G-box promoter element to modulate the expression of RD22 (responsive to desiccation 22), a drought/abscisic acid (ABA)-responsive gene (via a trans-expression QTL), and two strong yield-enhancement photosynthetic efficiency (PE) genes. This, in turn, upregulates other downstream drought-responsive and ABA signaling genes, as well as yield-enhancing PE genes, thus increasing plant adaptation to drought with reduced yield penalty. We showed that a superior allele of CabHLH10 introgressed into the NILs improved root and shoot biomass and PE, thereby enhancing yield and productivity during drought without compromising agronomic performance. Furthermore, overexpression of CabHLH10 in chickpea and Arabidopsis (Arabidopsis thaliana) conferred enhanced drought tolerance by improving root and shoot agro-morphological traits. These findings facilitate translational genomics for crop improvement and the development of genetically tailored, climate-resilient, high-yielding chickpea cultivars.

Cloning and function analysis of DlWRKY9 gene in longan (Dimocarpus longan)

WRKY is one of the largest plant transcription factors (TFs) which is widely involved in plant growth, development, and responses to stresses. In the present study, a WRKY TF DlWRKY9 was cloned from longan (Dimocarpus longan). The coding sequence (CDS) of DlWRKY9 is 762 bp in length and encodes 253 amino acids. It has a typical WRKY domain and zinc finger structure which belongs to type IIa WRKY protein. The molecular weight of DlWRKY9 protein was 30.27kda and the theoretical isoelectric point (PI) was 5.24. It is an unstable hydrophilic protein. The secondary structure of DlWRKY9 protein consists of helical structure (17.39%), extended chain (8.70%) and other structures (turn and random coil) (73.91%). The amino acid sequence of DlWRKY9 protein had the highest similarity with DlWRKY9 (xp_006450293.1) of citrus Clementina. DlWRKY9 gene promoter elements contain light, abscisic acid, gibberellin, jasmonic acid and other response elements. The results of qRT-PCR showed that the relative expression level of DlWRKY9 gene in pericarp was higher, followed by young fruits and floral organs. Meanwhile, DlWRKY9 gene specifically down-regulated in the early stage of flower induction in ‘Sijimi’ (SJ) longan. The results of transient expression of Arabidopsis protoplasts showed that the fluorescence signal was mainly concentrated in the nucleus. Moreover, overexpression of DlWRKY9 in Arabidopsis promoted early flowering. These results provide useful information for revealing the biological roles of DlWRKY9 in longan and increase our understanding of the WRKY family in fruit trees.

Promotional Elements in the "Me and Kajongan Tourism Village" Video Vlog

The city of Tegal is known as a maritime city, a city with a large fishing sector. The expanse of cultivators' ponds and mangrove trees provide a special beauty for the coastal villages in Tegal City. Kajongan tourism village is located at the west end of Muarareja Village, West Tegal District, Tegal City which is a potential fishery sector in Tegal City. In order to increase the enthusiasm of local and national communities to visit the Kajongan Tourism Village. The Regional Government through Bappeda organized the Kajongan Tourism Village Creative Video Competition “Come on Dolan Maring Kajongan”. The competition was held in the form of a video vlog as an effort to find the best video as a promotional medium for the Kajongan tourist village. The results of the competition were the 6 best videos in the general champion category which were selected based on the elements of promotion, video quality, story telling, shooting angles, and video editing. Ramadhan, aims to find promotional elements in the video vlog. data collection observations and interviews with informants The results of this study found elements of promotion and elements of cinematography. Based on the order of appearance, the abstract comprises the goal of the research, method implemented, results of the research, brief discussion, conclusion, and impact of the research.

APOE‐targeted epigenome therapy for late onset Alzheimer’s disease

AbstractBackgroundThere is an urgent need to refocus Alzheimer’s disease (AD) drug discovery on new targets and shifting the paradigm of AD DRUG DEVELOPMENT towards precision medicine. Apolipoprotein E gene (APOE) is the strongest and most reproduceable genetic risk factor for late‐onset Alzheimer’s disease (LOAD), and thus holds promise as a potential therapeutics target for LOAD. In this study we developed an epigenome therapy platform to reduce APOE expression generally and APOEe4 specifically by targeted modification of the epigenome landscape within APOE locus.MethodsOur gene therapy strategy is based on CRISPR/deactivated (d)Cas9 editing technology fused with an effector molecule and delivered by lentiviral (LV) vehicle. Our gRNAs were designed to target regulatory elements in the APOE promoter and in exon 4 overlapping the SNP that define the APOEe4 allele. We evaluated our epigenome therapy platform in vitro using human hiPSC‐derived neurons and in vivo by stereotactic injection of reporter gene into the hippocampus of mice.ResultsThe LV dCas9‐repressor vector showed decreased APOE‐mRNA and protein overall levels in hiPSC‐derived neuronal model. To specifically target the APOEe4 allele we utilized the VRER‐dCas9 protein. Evaluation of the system specificity showed a reduction in APOE‐mRNA levels in the hiPSC‐derived neurons with the e4 allele while there was no effect in the isogeneic hiPSC‐derived neurons homozygous for the e3 allele. Moving onto in vivo studies in mice administration of LV dCas9‐repressor vector and the GFP reporter gene into the hippocampus showed 50‐70% decrease in GFP expression demonstrating promising preliminary data. Collectively, our results provided in vitro and in vivo proof‐of‐concept for the utility and efficacy of the APOE‐targeted epigenome therapy.ConclusionsOur epigenome therapy strategy for fine‐tuning of APOE expression based on dCas9 technology is translational toward the development of a therapeutics approach to prevent and/or delay LOAD onset. Furthermore, the technology offers the opportunity to refine the platform for the development of gene‐specific and even allele‐ and cell‐type‐ specific therapies, and by that enables the advancement of strategies for precision medicine in LOAD.

Comprehensive in silico analysis of Phospholipase D gene family in economically important orchids

• A total of 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified. • These proteins are comprised of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. • The multiple sequence alignment, domain and motifs analysis revealed their conserved nature and homology structure simulation and Ramachadran plot analysis revealed the abundance of beta sheets in tertiary structures. • The presence of Root-specific element, DOF proteins binding domain, scaffold or matrix attachment region, pollen-specific, dehydration-responsive, light-responsive, gibberellin-responsive, ABA-responsive elements and Meja-responsive upstream cis- regulatory promoter elements indicated their role in various physiological processes. Phospholipase D (PLD) plays a fundamental role in various aspects of plant growth, development and stress responses. In present study, 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified on the basis of presence of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. These proteins were clustered into five sub-groups (α, β, δ, ζ and φ) along with their orthologs in Arabidopsis thaliana and Oryza sativa upon phylogenetic analysis. Gene characterization divulged that most of the PePLD, DcPLD and AsPLD genes within the same sub-group exhibited similar exon-intron organization and highly conserved motifs. RNA-seq based spatio-temporal studies indicated differential expression in various vegetative and reproductive tissues. PePLDα 1 , DcPLDα 1 and AsPLDα 1 were showing significant expression in all the tissues, while for other genes the expression was limited either in vegetative or reproductive tissues. Homology structure simulation revealed the abundance of random coils in secondary structures and beta sheets in tertiary structures. The presence of specific upstream cis- regulatory promoter elements emphasized the role of PLD s in various physiological processes. This report is the first genome-wide characterization of the PLD gene family in orchids and shall provide new insight into the functional characterization of these genes.

A CRISPRi-based investigation reveals that multiple promoter elements drive gene expression from the genome of mycobacteriophage D29.

A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus -35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.

Genome-wide identification, comprehensive characterization of transcription factors, cis-regulatory elements, protein homology, and protein interaction network of DREB gene family in Solanum lycopersicum.

Tomato is a drought-sensitive crop which has high susceptibility to adverse climatic changes. Dehydration-responsive element-binding (DREB) are significant plant transcription factors that have a vital role in regulating plant abiotic stress tolerance by networking with DRE/CRT cis-regulatory elements in response to stresses. In this study, bioinformatics analysis was performed to conduct the genome-wide identification and characterization of DREB genes and promoter elements in Solanum lycopersicum. In genome-wide coverage, 58 SlDREB genes were discovered on 12 chromosomes that justified the criteria of the presence of AP2 domain as conserved motifs. Intron-exon organization and motif analysis showed consistency with phylogenetic analysis and confirmed the absence of the A3 class, thus dividing the SlDREB genes into five categories. Gene expansion was observed through tandem duplication and segmental duplication gene events in SlDREB genes. Ka/Ks values were calculated in ortholog pairs that indicated divergence time and occurrence of purification selection during the evolutionary period. Synteny analysis demonstrated that 32 out of 58 and 47 out of 58 SlDREB genes were orthologs to Arabidopsis and Solanum tuberosum, respectively. Subcellular localization predicted that SlDREB genes were present in the nucleus and performed primary functions in DNA binding to regulate the transcriptional processes according to gene ontology. Cis-acting regulatory element analysis revealed the presence of 103 motifs in 2.5-kbp upstream promoter sequences of 58 SlDREB genes. Five representative SlDREB proteins were selected from the resultant DREB subgroups for 3D protein modeling through the Phyre2 server. All models confirmed about 90% residues in the favorable region through Ramachandran plot analysis. Moreover, active catalytic sites and occurrence in disorder regions indicated the structural and functional flexibility of SlDREB proteins. Protein association networks through STRING software suggested the potential interactors that belong to different gene families and are involved in regulating similar functional and biological processes. Transcriptome data analysis has revealed that the SlDREB gene family is engaged in defense response against drought and heat stress conditions in tomato. Overall, this comprehensive research reveals the identification and characterization of SlDREB genes that provide potential knowledge for improving abiotic stress tolerance in tomato.

Valproic Acid-Induced Upregulation of Multidrug Efflux Transporter ABCG2/BCRP via PPARα-Dependent Mechanism in Human Brain Endothelial Cells.

Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first- and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein, and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin, and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assays showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. SIGNIFICANCE STATEMENT: Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.