Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits.

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.

Genome-wide analysis of the NRAMP gene family in potato (Solanum tuberosum): Identification, expression analysis and response to five heavy metals stress.

NRAMP family genes participate in the absorption and transport of heavy metals such as cadmium (Cd), zinc (Zn), copper (Cu), lead (Pb), iron (Fe) and manganese (Mn) and play an important role in the response to heavy metal stress. There is an abundance of research on these genes in bacteria, plants and fungi, although not in S. tuberosum. A total of 48 members(potato(5), Arabidopsis(7), Tomato(9), pepper(9), rice(12) and tobacco(6)) were identified from 6 species (potato (Solanum tuberosum), Arabidopsis thaliana, Tomato (Solanum lycopersicum), pepper (Capsicum annuum), rice (Oryza sativa) and tobacco (Nicotiana attenuate)) and were classified into four subgroups. Across NRAMP gene family members, there are 15 highly conserved motifs that have similar genetic structures and characteristics. In addition, a total of 16 pairs of colinear genes were found in eight species. Analysis of cis-elements indicated that, in response to abiotic stress, NRAMPs are mainly regulated by phytohormones and transcription factors. In addition, analysis of expression profiles indicated that StNRAMP4 is mainly expressed in the roots. According to a qRT-PCR-based analysis of the StNRAMP family, with the exception of Pb2+ stress, StNRAMPs positively responded to stress from Cu2+, Cd2+, Zn2+ and Ni2+ and The expression patterns is similar of StNRAMP2, under Pb2+, and Cu2+ treatment, the relative expression peaked at 24 h. the relative expression peaked at 12 h and was upregulated 428-fold in the roots under Ni2+ stress. Under Cd2+ stress, StNRAMP3 was upregulated 28-fold in the leaves. StNRAMP1, StNRAMP4 and StNRAMP5 showed significant upregulation under Cu2+, Cd2+ and Zn2+ stress, respectively. Expression of StNRAMPs could be specifically induced by heavy metals, implying their possible role in the transport and absorption of heavy metals. This research explains the colinear characteristics of NRAMPs in several food crop species, which is useful for providing important genetic resources for cultivating food crop that accumulate low amounts of heavy metals and for explaining the biological functions of NRAMPs in plants.

Genome-Wide Characterization and Analysis of CIPK Gene Family in Two Cultivated Allopolyploid Cotton Species: Sequence Variation, Association with Seed Oil Content, and the Role of GhCIPK6.

Calcineurin B-like protein-interacting protein kinases (CIPKs), as key regulators, play an important role in plant growth and development and the response to various stresses. In the present study, we identified 80 and 78 CIPK genes in the Gossypium hirsutum and G. barbadense, respectively. The phylogenetic and gene structure analysis divided the cotton CIPK genes into five groups which were classified into an exon-rich clade and an exon-poor clade. A synteny analysis showed that segmental duplication contributed to the expansion of Gossypium CIPK gene family, and purifying selection played a major role in the evolution of the gene family in cotton. Analyses of expression profiles showed that GhCIPK genes had temporal and spatial specificity and could be induced by various abiotic stresses. Fourteen GhCIPK genes were found to contain 17 non-synonymous single nucleotide polymorphisms (SNPs) and co-localized with oil or protein content quantitative trait loci (QTLs). Additionally, five SNPs from four GhCIPKs were found to be significantly associated with oil content in one of the three field tests. Although most GhCIPK genes were not associated with natural variations in cotton oil content, the overexpression of the GhCIPK6 gene reduced the oil content and increased C18:1 and C18:1+C18:1d6 in transgenic cotton as compared to wild-type plants. In addition, we predicted the potential molecular regulatory mechanisms of the GhCIPK genes. In brief, these results enhance our understanding of the roles of CIPK genes in oil synthesis and stress responses.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes.

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Computational characterization of chromatin domain boundary-associated genomic elements.

Topologically associated domains (TADs) are 3D genomic structures with high internal interactions that play important roles in genome compaction and gene regulation. Their genomic locations and their association with CCCTC-binding factor (CTCF)-binding sites and transcription start sites (TSSs) were recently reported. However, the relationship between TADs and other genomic elements has not been systematically evaluated. This was addressed in the present study, with a focus on the enrichment of these genomic elements and their ability to predict the TAD boundary region. We found that consensus CTCF-binding sites were strongly associated with TAD boundaries as well as with the transcription factors (TFs) Zinc finger protein (ZNF)143 and Yin Yang (YY)1. TAD boundary-associated genomic elements include DNase I-hypersensitive sites, H3K36 trimethylation, TSSs, RNA polymerase II, and TFs such as Specificity protein 1, ZNF274 and SIX homeobox 5. Computational modeling with these genomic elements suggests that they have distinct roles in TAD boundary formation. We propose a structural model of TAD boundaries based on these findings that provides a basis for studying the mechanism of chromatin structure formation and gene regulation.

Allele-specific quantitative proteomics unravels molecular mechanisms modulated by cis-regulatory PPARG locus variation.

Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations.

Biochemical characterization of the selenoproteome in Gallus gallus via bioinformatics analysis: structure-function relationships and interactions of binding molecules.

Knowledge about mammalian selenoproteins is increasing. However, the selenoproteome of birds remains considerably less understood, especially concerning its biochemical characterization, structure-function relationships and the interactions of binding molecules. In this work, the SECIS elements, subcellular localization, protein domains and interactions of binding molecules of the selenoproteome in Gallus gallus were analyzed using bioinformatics tools. We carried out comprehensive analyses of the structure-function relationships and interactions of the binding molecules of selenoproteins, to provide biochemical characterization of the selenoproteome in Gallus gallus. Our data provided a wealth of information on the biochemical functions of bird selenoproteins. Members of the selenoproteome were found to be involved in various biological processes in chickens, such as in antioxidants, maintenance of the redox balance, Se transport, and interactions with metals. Six membrane-bound selenoproteins (SelI, SelK, SelS, SelT, DIO1 and DIO3) played important roles in maintaining the membrane integrity. Chicken selenoproteins were classified according to their ligand binding sites as zinc-containing matrix metalloselenoproteins (Sep15, MsrB1, SelW and SelM), POP-containing selenoproteins (GPx1-4), FAD-interacting selenoproteins (TrxR1-3), secretory transport selenoproteins (GPx3 and SelPa) and other selenoproteins. The results of our study provided new evidence for the unknown biological functions of the selenoproteome in birds. Future research is required to confirm the novel biochemical functions of bird selenoproteins.

Identification of a U/Zn/Cu responsive global regulatory two-component system in Caulobacter crescentus.

Despite the well-known toxicity of uranium (U) to bacteria, little is known about how cells sense and respond to U. The recent finding of a U-specific stress response in Caulobacter crescentus has provided a foundation for studying the mechanisms of U- perception in bacteria. To gain insight into this process, we used a forward genetic screen to identify the regulatory components governing expression of the urcA promoter (PurcA ) that is strongly induced by U. This approach unearthed a previously uncharacterized two-component system, named UzcRS, which is responsible for U-dependent activation of PurcA . UzcRS is also highly responsive to zinc and copper, revealing a broader specificity than previously thought. Using ChIP-seq, we found that UzcR binds extensively throughout the genome in a metal-dependent manner and recognizes a noncanonical DNA-binding site. Coupling the genome-wide occupancy data with RNA-seq analysis revealed that UzcR is a global regulator of transcription, predominately activating genes encoding proteins that are localized to the cell envelope; these include metallopeptidases, multidrug-resistant efflux (MDR) pumps, TonB-dependent receptors and many proteins of unknown function. Collectively, our data suggest that UzcRS couples the perception of U, Zn and Cu with a novel extracytoplasmic stress response.

Genome-wide patterns of regulatory divergence revealed by introgression lines.

Understanding the genetic basis for changes in transcriptional regulation is an important aspect of understanding phenotypic evolution. Using interspecific introgression lines, we infer the mechanisms of divergence in genome-wide patterns of gene expression between the nightshades Solanum pennellii and S. lycopersicum (domesticated tomato). We find that cis- and trans-regulatory changes have had qualitatively similar contributions to divergence in this clade, unlike results from other systems. Additionally, expression data from four tissues (shoot apex, ripe fruit, pollen, and seed) suggest that introgressed regions in these hybrid lines tend to be downregulated, while background (nonintrogressed) genes tend to be upregulated. Finally, we find no evidence for an association between the magnitude of differential expression in NILs and previously determined sterility phenotypes. Our results contradict previous predictions of the predominant role of cis- over trans-regulatory divergence between species, and do not support a major role for gross genome-wide misregulation in reproductive isolation between these species.

Transcriptional regulation of Tal2 gene by all-trans retinoic acid (atRA) in P19 cells.

TAL2 is a transcription factor required in the normal development of mouse brain. In a previous study, we demonstrated that the expression of Tal2 gene is induced by the complex of all-trans retinoic acid (atRA) and retinoic acid receptor α (RARα) in mouse embryonal carcinoma P19 cells. atRA is also known to be important in inducing P19 cells to differentiate into the neural lineage. Therefore, we believe that the function of TAL2 in neural differentiation may be clarified by utilizing P19 cells. As the atRA-RARα complex induced the expression of Tal2, we focused on the regulatory region that is involved in its transcription. The atRA-RARα complex occupies a characteristic retinoic acid response element (RARE) located in the promoter of target genes. Therefore, we searched for RARE on the mouse Tal2 and found that a RARE-like element was located in the intron. We also found that a TATA-box-like element was located in the 5'-region of Tal2. Involvement between transcriptional activity and the TATA-box-like element was confirmed in the luciferase assay, and TATA-box binding protein was bound to this element upstream of Tal2 in P19 cells. atRA signaling activated the transcription through the RARE-like element, and RARα was bound to this element on Tal2 in P19 cells. In addition, the interaction between these elements on Tal2 was shown in the chromatin immunoprecipitation assay. These results suggest that the transcription of Tal2 is coordinately mediated by two distal regulatory elements.

Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops.

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding ß-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.

De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism.

Centella asiatica (L.) Urban is an important medicinal plant and has been used since ancient times in traditional systems of medicine. C. asiatica mainly contains ursane skeleton based triterpenoid sapogenins and saponins predominantly in its leaves. This investigation employed Illumina next generation sequencing (NGS) strategy on a pool of three cDNAs from expanding leaf of C. asiatica and developed an assembled transcriptome sequence resource of the plant. The short transcript reads (STRs) generated and assembled into contigs and singletons, representing majority of the genes expressed in C. asiatica, were termed as 'tentative unique transcripts' (TUTs). The TUT dataset was analyzed with the objectives of (i) development of a transcriptome assembly of C. asiatica, and (ii) classification/characterization of the genes into categories like structural, functional, regulatory etc. based on their function. Overall, 68.49% of the 46,171,131 reads generated in the NGS process could be assembled into a total of 79,041 contigs. Gene ontology and functional annotation of sequences resulted into the identification of genes related to different sets of cellular functions including identification of genes related to primary and secondary metabolism. The wet lab validation of seventeen assembled gene sequences identified to be involved in secondary metabolic pathways and control of reactive oxygen species (ROS) was established by semi-quantitative and real time PCR (qRT-PCR). The validation also included sequencing/size matching of a set of semi-quantitative PCR amplicons with their in silico assembled contig/gene. This confirmed the appropriateness of assembling the reads and contigs. Thus, the present study constitutes the largest report to date on C. asiatica transcriptome based gene resource that may contribute substantially to the understanding of the basal biological functions and biochemical pathways of secondary metabolites as well as the transcriptional regulatory elements and genetic markers. This work sets the stage for multi-faceted future improvement of the plant, through discovery of new genes, marker-assisted breeding or genetic engineering, on this species as well as for other species of Apiaceae and triterpene producing medicinal plants.

Regulatory elements associated with paternally-expressed genes in the imprinted murine Angelman/Prader-Willi syndrome domain.

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain - such as MKRN3 and NDN - are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.

Identification of causal genes, networks, and transcriptional regulators of REM sleep and wake.

STUDY OBJECTIVE: Sleep-wake traits are well-known to be under substantial genetic control, but the specific genes and gene networks underlying primary sleep-wake traits have largely eluded identification using conventional approaches, especially in mammals. Thus, the aim of this study was to use systems genetics and statistical approaches to uncover the genetic networks underlying 2 primary sleep traits in the mouse: 24-h duration of REM sleep and wake. DESIGN: Genome-wide RNA expression data from 3 tissues (anterior cortex, hypothalamus, thalamus/midbrain) were used in conjunction with high-density genotyping to identify candidate causal genes and networks mediating the effects of 2 QTL regulating the 24-h duration of REM sleep and one regulating the 24-h duration of wake. SETTING: Basic sleep research laboratory. PATIENTS OR PARTICIPANTS: Male [C57BL/6J × (BALB/cByJ × C57BL/6J*) F1] N(2) mice (n = 283). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The genetic variation of a mouse N2 mapping cross was leveraged against sleep-state phenotypic variation as well as quantitative gene expression measurement in key brain regions using integrative genomics approaches to uncover multiple causal sleep-state regulatory genes, including several surprising novel candidates, which interact as components of networks that modulate REM sleep and wake. In particular, it was discovered that a core network module, consisting of 20 genes, involved in the regulation of REM sleep duration is conserved across the cortex, hypothalamus, and thalamus. A novel application of a formal causal inference test was also used to identify those genes directly regulating sleep via control of expression. CONCLUSION: Systems genetics approaches reveal novel candidate genes, complex networks and specific transcriptional regulators of REM sleep and wake duration in mammals.

The anaphase-promoting complex is a dual integrator that regulates both MicroRNA-mediated transcriptional regulation of cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development.

The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by DUO POLLEN1 (DUO1). DUO1 is required for cell division in male gametophytes and is a target of microRNA 159 (miR159) in Arabidopsis thaliana. Whether APC/C is required for DUO1-dependent CYCB1;1 regulation is unknown. Mutants in both APC8 and APC13 had pleiotrophic phenotypes resembling those of mutants affecting microRNA biogenesis. We show that these apc/c mutants had reduced miR159 levels and increased DUO1 and CYCB1;1 transcript levels and that APC/C is required to recruit RNA polymerase II to MIR159 promoters. Thus, in addition to its role in degrading CYCB1;1, APC/C stimulates production of miR159, which downregulates DUO1 expression, leading to reduced CYCB1;1 transcription. Both MIR159 and APC8-yellow fluorescent protein accumulated in unicellular microspores and bicellular pollen but decreased in tricellular pollen, suggesting that spatial and temporal regulation of miR159 by APC/C ensures mitotic progression. Consistent with this, the percentage of mature pollen with no or single sperm-like cells increased in apc/c mutants and plants overexpressing APC8 partially mimicked the duo1 phenotype. Thus, APC/C is an integrator that regulates both microRNA-mediated transcriptional regulation of CYCB1;1 and degradation of CYCB1;1.

Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-ß gene.

Transcriptional regulation has a critical role in the coordinate and context-specific expression of a cluster of genes encoding members of the tumour necrosis factor (TNF) superfamily found at chromosome 6p21, comprising TNF, LTA (encoding lymphotoxin-α) and LTB (encoding lymphotoxin-ß). This is important, as dysregulated expression of these genes is implicated in susceptibility to many autoimmune, inflammatory and infectious diseases. We describe here a novel regulatory element in the fourth exon of LTB, which is highly conserved, localises to the only CpG island in the locus, and is associated with a DNase I hypersensitive site and specific histone modifications. We find evidence of binding by Yin Yang 1 (YY1), cyclic AMP response element (CRE)-binding protein (CREB) and CCCTC-binding factor (CTCF) to this region in Jurkat T cells, which is associated with transcriptional repression on reporter gene analysis. Chromatin conformation capture experiments show evidence of DNA looping, involving interaction of this element with the LTB promoter, LTA promoter and TNF 3' untranslated region (UTR). Small interfering RNA (siRNA) experiments demonstrate a functional role for YY1 and CREB in LTB expression. Our findings provide evidence of additional complexity in the transcriptional regulation of LTB with implications for coordinate expression of genes in this important genomic locus.

Pollen-specific expression of Oryza sativa indica pollen allergen gene (OSIPA) promoter in rice and Arabidopsis transgenic systems.

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter--upon expression in tobacco and Arabidopsis--was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5' deletions were fused to ß-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to -1272, -966, -617, and -199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between -1567 and -199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.

Molecular cloning and characterization of an achene-seed-specific promoter from motherwort (Leonurus japonicus Houtt).

LJAMP1 is a small antimicrobial protein purified previously from the seeds of motherwort, and it is expressed preferentially in seeds. A 794-bp upstream sequence of the ATG start codon was isolated using a genome walking method and cloned into the upstream of the ß-glucuronidase (GUS) reporter gene to determine the GUS tissue-specific expression pattern. The transgenic tobacco showed that pLJAMP1 promoter derived GUS reporter gene special expression in pollen, achene and seed. The analysis of cis-acting elements also revealed pLJAMP1 promoter contained pollen and seed related transcriptional control elements.

A negative element in the downstream region of the Rice tungro bacilliform virus promoter is orientation- and position-independent and is active with heterologous promoters.

The promoter of an Indian isolate of the pararetrovirus Rice tungro bacilliform virus (RTBV-WB) contains a negative element downstream of the transcription start site (TSS), between nucleotide residues +58 and +195 (Mathur and Dasgupta, 2007). To further characterize the element, we show, by using transient gus reporter gene assays in the cells of onion peel, rice calli and Arabidopsis leaves, that it down-regulates heterologous promoters CaMV35S and Maize ubiquitin. Quantitative measurements of transient GUS activity indicated more than 90% inhibition of reporter gene expression by the negative element. We also show, by reversing the orientation of the element downstream and by placing it in a position upstream to a constitutively expressing RTBV promoter, that the negative element is orientation- and position-independent, pointing towards its activity at the transcriptional and not post-transcriptional level.