m 6 Aexpress-BHM: predicting m6A regulation of gene expression in multiple-groups context by a Bayesian hierarchical mixture model.

As the most abundant RNA modification, N6-methyladenosine (m6A) plays an important role in various RNA activities including gene expression and translation. With the rapid application of MeRIP-seq technology, samples of multiple groups, such as the involved multiple viral/ bacterial infection or distinct cell differentiation stages, are extracted from same experimental unit. However, our current knowledge about how the dynamic m6A regulating gene expression and the role in certain biological processes (e.g. immune response in this complex context) is largely elusive due to lack of effective tools. To address this issue, we proposed a Bayesian hierarchical mixture model (called m6Aexpress-BHM) to predict m6A regulation of gene expression (m6A-reg-exp) in multiple groups of MeRIP-seq experiment with limited samples. Comprehensive evaluations of m6Aexpress-BHM on the simulated data demonstrate its high predicting precision and robustness. Applying m6Aexpress-BHM on three real-world datasets (i.e. Flaviviridae infection, infected time-points of bacteria and differentiation stages of dendritic cells), we predicted more m6A-reg-exp genes with positive regulatory mode that significantly participate in innate immune or adaptive immune pathways, revealing the underlying mechanism of the regulatory function of m6A during immune response. In addition, we also found that m6A may influence the expression of PD-1/PD-L1 via regulating its interacted genes. These results demonstrate the power of m6Aexpress-BHM, helping us understand the m6A regulatory function in immune system.

Three new LmbU targets outside lmb cluster inhibit lincomycin biosynthesis in Streptomyces lincolnensis.

BACKGROUND: Antibiotics biosynthesis is usually regulated by the cluster-situated regulatory gene(s) (CSRG(s)), which directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). Previously, we have demonstrated that LmbU functions as a cluster-situated regulator (CSR) of lincomycin. And it has been found that LmbU regulates twenty non-lmb genes through comparative transcriptomic analysis. However, the regulatory mode of CSRs' targets outside the BGC remains unknown. RESULTS: We screened the targets of LmbU in the whole genome of Streptomyces lincolnensis and found fourteen candidate targets, among which, eight targets can bind to LmbU by electrophoretic mobility shift assays (EMSA). Reporter assays in vivo revealed that LmbU repressed the transcription of SLINC_0469 and SLINC_1037 while activating the transcription of SLINC_8097. In addition, disruptions of SLINC_0469, SLINC_1037, and SLINC_8097 promoted the production of lincomycin, and qRT-PCR showed that SLINC_0469, SLINC_1037, and SLINC_8097 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. CONCLUSIONS: LmbU can directly regulate genes outside the lmb cluster, and these genes can affect both lincomycin biosynthesis and the transcription of lmb genes. Our results first erected the cascade regulatory circuit of LmbU and regulators outside lmb cluster, which provides the theoretical basis for the functional research of LmbU family proteins.

Production improvement of an antioxidant in cariogenic Streptococcus mutans UA140.

AIMS: This study aimed to improve the production of mutantioxidin, an antioxidant encoded by a biosynthetic gene cluster (mao) in Streptococcus mutans UA140, through a series of optimization methods. METHOD AND RESULTS: Through the construction of mao knockout strain S. mutans UA140∆mao, we identified mutantioxidin as the antioxidant encoded by mao and verified its antioxidant activity through a reactive oxygen species (ROS) tolerance assay. By optimizing the culture medium and fermentation time, 72 h of fermentation in chemically defined medium (CDM) medium was determined as the optimal fermentation conditions. Based on two promoters commonly used in Streptococcus (ldhp and xylS1p), eight promoter refactoring strains were constructed, nevertheless all showed impaired antioxidant production. In-frame deletion and complementation experiments demonstrated the positive regulatory role of mao1 and mao2, on mao. Afterward, the mao1 and mao2, overexpression strain S. mutans UA140/pDL278:: mao1mao2, were constructed, in which the production of mutantioxidin was improved significantly. CONCLUSIONS: In this study, through a combination of varied strategies such as optimization of fermentation conditions and overexpression of regulatory genes, production of mutantioxidin was increased by 10.5 times ultimately.

Identification and Analysis of lncRNA and circRNA Related to Wheat Grain Development.

The role of lncRNA and circRNA in wheat grain development is still unclear. The objectives of this study were to characterize the lncRNA and circRNA in the wheat grain development and to construct the interaction network among lncRNA, circRNA, and their target miRNA to propose a lncRNA-circRNA-miRNA module related to wheat grain development. Full transcriptome sequencing on two wheat varieties (Annong 0942 and Anke 2005) with significant differences in 1000-grain weight at 10 d (days after pollination), 20 d, and 30 d of grain development were conducted. We detected 650, 736, and 609 differentially expressed lncRNA genes, and 769, 1054, and 1062 differentially expressed circRNA genes in the grains of 10 days, 20 days and 30 days after pollination between Annong 0942 and Anke 2005, respectively. An analysis of the lncRNA-miRNA and circRNA-miRNA targeting networks reveals that circRNAs exhibit a more complex and extensive interaction network in the development of cereal grains and the formation of grain shape. Central to these interactions are tae-miR1177, tae-miR1128, and tae-miR1130b-3p. In contrast, lncRNA genes only form a singular network centered around tae-miR1133 and tae-miR5175-5p when comparing between varieties. Further analysis is conducted on the underlying genes of all target miRNAs, we identified TaNF-YB1 targeted by tae-miR1122a and TaTGW-7B targeted by miR1130a as two pivotal regulatory genes in the development of wheat grains. The quantitative real-time PCR (qRT-PCR) and dual-luciferase reporter assays confirmed the target regulatory relationships between miR1130a-TaTGW-7B and miR1122a-TaNF-YB1. We propose a network of circRNA and miRNA-mediated gene regulation in the development of wheat grains.

Screening for brown-spot disease and drought stress response and identification of dual-stress responsive genes in rice cultivars of Northeast India.

Rice cultivation in Northeast India (NEI) primarily relies on rainfed conditions, making it susceptible to severe drought spells that promote the onset of brown spot disease (BSD) caused by Bipolaris oryzae. This study investigates the response of prevalent rice cultivars of NEI to the combined stress of drought and B. oryzae infection. Morphological, physiological, biochemical, and molecular changes were recorded post-stress imposition. Qualitative assessment of reactive oxygen species through DAB (3,3-diaminobenzidine) assay confirmed the elicitation of plant defense responses. Based on drought scoring system and biochemical analyses, the cultivars were categorized into susceptible (Shasharang and Bahadur), moderately susceptible (Gitesh and Ranjit), and moderately tolerant (Kapilee and Mahsuri) groups. Antioxidant enzyme accumulation (catalase, guaiacol peroxidase) and osmolyte (proline) levels increased in all stressed plants, with drought-tolerant cultivars exhibiting higher enzyme activities, indicating stress mitigation efforts. Nevertheless, electrolyte leakage and lipid peroxidation rates increased in all stressed conditions, though variations were observed among stress types. Based on findings from a previous transcriptomic study, a total of nine genes were chosen for quantitative real-time PCR analysis. Among these, OsEBP89 appeared as a potential negative regulatory gene, demonstrating substantial upregulation in the susceptible cultivars at both 48 and 72 h post-treatment (hpt). This finding suggests that OsEBP89 may play a role in conferring drought-induced susceptibility to BSD in the rice cultivars being investigated. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01447-4.

ETS1 regulates NDRG1 to promote the proliferation, migration, and invasion of OSCC.

OBJECTIVE: The aim of this study was to investigate the molecular mechanism by which the transcription factor ETS1 regulates N-myc downstream regulatory gene 1 (NDRG1) to provide a new theoretical basis for the study of oral squamous cell carcinoma (OSCC). METHODS: In this study, eight human OSCC and paraneoplastic samples were collected. The expressions of NDRG1, ETS1, and Ki67 were detected by immunohistochemistry; apoptosis was detected by tdt-mediated dUTP notched end labeling; cell migration and invasion were detected by Transwell; quantitative real-time PCR was performed to detect the expression of NDRG1; RNA-binding protein immunoprecipitation (RIP) assays detected NDRG1 expression; immunofluorescence assays detected ETS1 expression. RESULTS: NDRG1 and ETS1 expression was significantly upregulated in cancer tissues and CAL-27 and SCC-6 cells. Knockdown of NDRG1 and ETS1 inhibited cell proliferation, migration, invasion, cloning, and EMT while promoting apoptosis and inhibited tumor development; ETS1 positively regulated NDRG1 expression; Finally, overexpression of NDRG1 in vivo and in vitro reversed the effect of ETS1 knockdown on CAL-27 and SCC-6 cells. CONCLUSIONS: ETS1 positively regulates the expression of NDRG1 and promotes OSCC. Therefore, ETS1 may serve as a new target for the clinical diagnosis and treatment of OSCC.

SlyA regulates virulence gene expressions through activation of pchA regulatory gene in enterohemorrhagic Escherichia coli.

SlyA is a DNA-binding protein that alters the nucleoid complex composed of histone-like nucleoid-structuring protein (H-NS) and activates gene expression. In enterohemorrhagic Escherichia coli (EHEC), the expression of virulence genes is repressed by H-NS but is up-regulated in response to environmental factors by releasing a nucleoid complex. This study examined the effect of slyA deletion mutation in EHEC and discovered that the production of the locus of enterocyte effacement (LEE)-encoded EspB and Tir, as well as the cell adherence ability, was reduced in the mutant compared with the wild type. The promoter activity of the LEE1 operon, including the regulatory gene, ler, was reduced by slyA mutation, but tac promoter-controlled expression of pchA, which is a regulatory gene of LEE1, abolished the effect. The promoter activity of pchA was down-regulated by the slyA mutation. Furthermore, the coding region was required for its regulation and was bound to SlyA, which indicates the direct regulation of pchA by SlyA. However, the slyA mutation did not affect the butyrate-induced increase in pchA promoter activity. Additionally, the pchA promoter activity was increased via induction of lrp, a regulatory gene for butyrate response, in the slyA mutant and, conversely, by introducing high copies of slyA into the lrp mutant. These results indicate that SlyA is a positive regulator of pchA and is independent of the Lrp regulatory system. SlyA may be involved in the virulence expression in EHEC, maintaining a certain level of expression in the absence of a butyrate response.

Krüppel-like Transcription Factor 7 Is a Causal Gene in Autism Development.

BACKGROUND: Autism spectrum disorder (ASD) is a complex neurodevelopmental disease. To date, more than 1000 genes have been shown to be associated with ASD, and only a few of these genes account for more than 1% of autism cases. Klf7 is an important transcription factor of cell proliferation and differentiation in the nervous system, but whether klf7 is involved in autism is unclear. METHODS: We first performed ChIP-seq analysis of klf7 in N2A cells, then performed behavioral tests and RNA-seq in klf7+/- mice, and finally restored mice with adeno-associated virus (AAV)-mediated overexpression of klf7 in klf7+/- mice. RESULTS: Klf7 targeted genes are enriched with ASD genes, and 631 ASD risk genes are also differentially expressed in klf7+/- mice which exhibited the core symptoms of ASD. When klf7 levels were increased in the central nervous system (CNS) in klf7+/- adult mice, deficits in social interaction, repetitive behavior and majority of dysregulated ASD genes were rescued in the adults, suggesting transcriptional regulation. Moreover, knockdown of klf7 in human brain organoids caused dysregulation of 517 ASD risk genes, 344 of which were shared with klf7+/- mice, including some high-confidence ASD genes. CONCLUSIONS: Our findings highlight a klf7 regulation of ASD genes and provide new insights into the pathogenesis of ASD and promising targets for further research on mechanisms and treatments.

Regulatory and Enterotoxin Gene Expression and Enterotoxins Production in Staphylococcus aureus FRI913 Cultures Exposed to a Rotating Magnetic Field and trans-Anethole.

The study aimed to examine the influence of a rotating magnetic field (RMF) of two different frequencies (5 and 50 Hz) on the expression of regulatory (agrA, hld, rot) and staphylococcal enterotoxin (SE-sea, sec, sel) genes as well as the production of SEs (SEA, SEC, SEL) by the Staphylococcus aureus FRI913 strain cultured on a medium supplemented with a subinhibitory concentration of trans-anethole (TA). Furthermore, a theoretical model of interactions between the bacterial medium and bacterial cells exposed to RMF was proposed. Gene expression and SEs production were measured using quantitative real-time PCR and ELISA techniques, respectively. Based on the obtained results, it was found that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes expression as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SEs and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none.

Engineering Bafilomycin High-Producers by Manipulating Regulatory and Biosynthetic Genes in the Marine Bacterium Streptomyces lohii.

Bafilomycin A1 is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H+-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (baf) from the marine bacterium Streptomyces lohii ATCC BAA-1276, wherein a luxR family regulatory gene orf1 and an afsR family regulatory gene bafG were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of orf1 and bafG for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type S. lohii strain, the knockout of either orf1 or bafG completely abolished the production of bafilomycins. The overexpression of orf1 or bafG led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically engineered S. lohii strain (SLO-08) with orf1 overexpression and inactivation of the biosynthetic genes orf2 and orf3, solely produced bafilomycin A1 with the titer of 535.1 ± 25.0 mg/L in an optimized fermentation medium in shaking flasks. This recombinant strain holds considerable application potential in large-scale production of bafilomycin A1 for new drug development.

Senescence-Associated Glycine max (Gm)NAC Genes: Integration of Natural and Stress-Induced Leaf Senescence.

Leaf senescence is a genetically regulated developmental process that can be triggered by a variety of internal and external signals, including hormones and environmental stimuli. Among the senescence-associated genes controlling leaf senescence, the transcriptional factors (TFs) comprise a functional class that is highly active at the onset and during the progression of leaf senescence. The plant-specific NAC (NAM, ATAF, and CUC) TFs are essential for controlling leaf senescence. Several members of Arabidopsis AtNAC-SAGs are well characterized as players in elucidated regulatory networks. However, only a few soybean members of this class display well-known functions; knowledge about their regulatory circuits is still rudimentary. Here, we describe the expression profile of soybean GmNAC-SAGs upregulated by natural senescence and their functional correlation with putative AtNAC-SAGs orthologs. The mechanisms and the regulatory gene networks underlying GmNAC081- and GmNAC030-positive regulation in leaf senescence are discussed. Furthermore, new insights into the role of GmNAC065 as a negative senescence regulator are presented, demonstrating extraordinary functional conservation with the Arabidopsis counterpart. Finally, we describe a regulatory circuit which integrates a stress-induced cell death program with developmental leaf senescence via the NRP-NAC-VPE signaling module.

Transferrin receptor 1 overexpression is associated with tumour de-differentiation and acts as a potential prognostic indicator of hepatocellular carcinoma.

AIM: Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality worldwide. An excess of iron in liver tissue causes oxidative stress, leading to hepatocellular carcinogenesis. Iron metabolism, which is regulated by a complex mechanism, is important for cancer cell survival. The aim of this study is to clarify the role of iron regulatory protein in the progression of HCC and in patient outcome. METHODS AND RESULTS: We first investigated the mRNA level of iron metabolism-related genes, including hepcidin, ferroportin 1 (FPN-1) and transferrin receptor (TFR)-1/2. TFR-1/2 protein expression was then evaluated in surgical specimens from 210 cases using immunohistochemistry, and we compared clinicopathological factors with TFR-1/2 expression. The mRNA expression levels of TFR-1 were significantly increased in HCC tissues compared with adjacent non-cancerous tissues (P = 0.0013), but there were no differences in other genes. High expression of TFR-1 in HCC was associated with the absence of alcohol abuse (P = 0.0467), liver cirrhosis (P < 0.0001), higher alpha-fetoprotein (AFP; P < 0.0001), smaller tumour size (P = 0.0022), poor histological differentiation (P < 0.0001) and morphological features (P < 0.0001). In contrast, high expression of TFR-2 in HCC was associated with lower AFP (P < 0.0001), well-differentiated histological grade (P < 0.0001) and morphological features (P = 0.0010). Multivariate analysis for both overall survival and recurrence-free survival indicated that high TFR-1 expression was a significant prognostic factor for poor outcome. CONCLUSIONS: We found an inverse correlation of TFR-1 and TFR-2 expression in AFP and tumour differentiation. TFR-1 overexpression suggests a higher risk of recurrence and death in HCC patients following liver resection.

Pseudonocardia strain improvement for stimulation of the di-sugar heptaene Nystatin-like Pseudonocardia polyene B1 biosynthesis.

Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N'-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.

Development of fluorescent protein-based biosensing strains: A new tool for the detection of aromatic hydrocarbon pollutants in the environment.

The major sources for release of hydrocarbons into the environment include the effluents generated from chemical processing industries and ports. The introduction of such hazardous compounds into natural water bodies creates considerable disturbances in aquatic life and causes a threat to humans. Thus, it is essential to detect and quantify pollutants at various stages of the wastewater generation and treatment before they reach natural aquatic environments and contaminate them. This study reports the development of "biosensing strains" by cloning hydrocarbon recognizing promoter-operator and a reporter gene in bacterial strains for sensing the presence of pollutants at their lowest possible concentration. So far, various biosensing strains have been constructed with a fused promoter-operator region of the hydrocarbon degrading operons, but most of them use luxAB as a reporter gene. A novel approach in the present study aimed at constructing strains harboring two different fluorescent protein (FP)-based reporter genes for the quantification of multiple pollutants at a time. Two vectors were designed with a fusion of tbuT-gfp and phnR-cfp for the quantification of mono- and poly-aromatic hydrocarbons, respectively. The designed vectors were transformed into E. coli DH5α, and these strains were designated as E. coli DH5α 2296-gfp (containing pPROBE-Tbut-RBS-gfp-npt) and E. coli DH5α 2301-cfp (containing pPROBE-phn-RBS-cfp-npt). Both the developed recombinant strains were capable of successfully detecting mono- and poly-aromatic hydrocarbons in the range of 1-100 µM. The sensing capacity of recombinant strains was successfully validated with actual wastewater samples against available physico-chemical analytical techniques. The development of such recombinant microbial strains indicates the future for online contaminant detection, treatment quality monitoring and protection of aquatic flora and fauna.

Synergetic and Antagonistic Molecular Effects Mediated by the Feedback Loop of p53 and JNK between Saikosaponin D and SP600125 on Lung Cancer A549 Cells.

We jointly analyzed the changes in cell cycle arrest and distribution, the accumulation of subphase cells, apoptosis, and proliferation in A549 cells treated with Saikosaponin D (Ssd) and JNK inhibitor SP600125 alone or in combination. Our results indicated that cell cycle arrest at G0/G1, S, and G2/M phases was coupled with the accumulation of subG1, subS, and subG2 cells, corresponding to early apoptosis, DNA endoreplication, and later inhibitory proliferation, respectively. Analyzing the expression of 18 cell cycle regulatory genes and JNK and phosphorylated JNK (pJNK) levels revealed an enhancement in these factors by Ssd. Additional SP600125 weakened or eliminated the Ssd-induced increase of these factors except that p53/p21 and Rassfia levels were further improved. Ingenuity Pathway Analysis (IPA) of the interactions of these factors revealed a negative synergistic effect on apoptosis while a positive synergistic effect on proliferative inhibition of the two drugs: (1) Ssd induced apoptosis via the activation of two axes, TGFα-JNK-p53 and TGFα-Rassfia-Mst1. By eliminating the Ssd-induced increase of JNK/pJNK, additional SP600125 weakened the Ssd-induced apoptotic axis of TGFα-JNK-p53 and simultaneously abolished Ssd-induced apoptosis; (2) Ssd inhibited proliferation by the activation of two axes, TGFß-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1. By improving the Ssd-induced increase of p53/p21 and Rassfia, additional SP600125 enhanced the two axes of Ssd-induced inhibitory proliferation. Analyzing JNK/pJNK, p53, phospho-p53, and TNF-α levels revealed an opposite association of JNK/pJNK with p53 while consistent with phospho-p53 and TNF-α, which supported the proposals that JNK/pJNK negatively regulated p53 level, while it mediated p53 phosphorylation to transcriptionally activate TNF-α expression of apoptotic gene and trigger apoptosis. With the multiple roles, JNK/pJNK forms a synergetic and antagonistic feedback loop with phospho-p53/p53. Within the feedback loop, (1) Ssd-induced apoptosis depended on JNK/pJNK activities mediating phospho-p53 that activated TNF-α expression; (2) by weakening the negative regulation of JNK/pJNK in p53, SP600125 enhanced p53 level and the Ssd-induced inhibitory proliferation axes of TGFß-p53/p21/p27/p15/p16. The results indicated the central coordinating roles of the feedback loop in the synergistic and antagonistic effects of the two drugs in A549 cells and provided a rationale for the combination of Ssd with SP600125 in the treatment of lung cancer.

Complex regulation of the TaMyc1 gene expression in wheat grain synthesizing anthocyanin pigments.

The wheat TaMyc1 gene encodes for transcriptional factor (TF) with bHLH domain. The gene is expressed in purple wheat grains and activates transcription of the anthocyanin biosynthesis structural genes. To reveal the features of TaMyc1 regulation in wheat pericarp transcription start sites (TSS) were identified by 5' RACE mean and translation efficiency was predicted by in silico methods. Three alternative transcript variants of TaMyc1 differing by 5' leader sequence only were identified in purple pericarp. The three transcripts are generated from distinct TATA boxes and thereby are differed by TSS. Two transcripts (TaMyc1a, -b) have identical initiation AUG codons that lead to the TaMYC1 regulatory protein with bHLH domain. However because of different stability of secondary structures predicted in 5' leader the two transcripts might be translated with different efficiency. The third transcript is assumed to be not effectively translated. qRT-PCR and colonies counting were applied to assess contribution each of the transcripts to total TaMyc1 gene transcription level. TaMyc1c has the lowest contribution (ca. 16%), whereas the others two transcripts contribute equally (ca. 42%) to total TaMyc1 expression level. The role of the tree mRNA isoforms transcribed in one tissue is discussed.

Global analysis of the MATE gene family of metabolite transporters in tomato.

BACKGROUND: Species in the Solanaceae family are known for producing plethora of specialized metabolites. In addition to biosynthesis pathways, a full comprehension of secondary metabolism must also take into account the transport and subcellular compartmentalization of substances. Here, we examined the MATE (Multidrug and Toxic Compound Extrusion, or Multi-Antimicrobial Extrusion) gene family in the tomato (Solanum lycopersicum) genome with the objective of better understanding the transport of secondary metabolites in this model species. MATE membrane effluxers encompass an ancient gene family of secondary transporters present in all kingdoms of life, but with a remarkable expansion in plants. They mediate the transport of primary and secondary metabolites using the proton motive force through several membrane systems of the cell. RESULTS: We identified 67 genes coding for MATE transporters in the tomato genome, 33 of which are expressed constitutively whereas 34 are expressed in specific cell types or environmental conditions. Synteny analyses revealed bona fide paralogs and Arabidopsis orthologs. Co-expression analysis between MATE and regulatory genes revealed 78 positive and 8 negative strong associations (ρ≥|0.8|). We found no evidence of MATE transporters belonging to known metabolic gene clusters in tomato. CONCLUSIONS: Altogether, our expression data, phylogenetic analyses, and synteny study provide strong evidence of functional homologies between MATE genes of tomato and Arabidopsis thaliana. Our co-expression study revealed potential transcriptional regulators of MATE genes that warrant further investigation. This work sets the stage for genome-wide functional analyses of MATE transporters in tomato and other Solanaceae species of economic relevance.

Overexpression of biologically safe Rorippa indica defensin enhances aphid tolerance in Brassica juncea.

MAIN CONCLUSION: Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71-2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest-L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections.

In this study, we analyzed 15 multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa from chronic lung infections for expression of 4 different multidrug efflux systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY), using quantitative reverse transcriptase PCR. Overexpression of MexXY pump was observed in all of the isolates tested. Analysis of regulatory genes that control the expression of these 4 efflux pumps revealed a number of previously uncharacterized mutations. Our work shows that MexXY pump overexpression is common in cystic fibrosis isolates and could be contributing to their reduced aminoglycoside susceptibility. Further, we also identified novel mutations in the regulatory genes of the 4 abovementioned Resistance-Nodulation-Division superfamily pumps that may be involved in the overexpression of these pumps.

A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.

The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual ß-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo.