Effect of 5′UTR introns on gene expression in Arabidopsis thaliana

Background The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5′UTRs and 3′UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome. Results A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5′UTRs is similar to that in CDSs but much higher than that in 3′UTRs; (2) the 5′UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5′UTRs are, on average, longer than introns in the CDSs and 3′UTRs; and (4) 5′UTR introns have a different nucleotide composition to that of CDs and 3′UTR introns. Furthermore, we show that the 5′UTR intron of the A. thaliana EFIα-A3 gene affects the gene expression and the size of the 5′UTR intron influences the level of gene expression. Conclusion Introns within the 5′UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3′UTR. In the EFIα-A3 gene, the presence of a long intron in the 5′UTR is sufficient to enhance gene expression in plants in a size dependent manner.

Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Genetic aspects of the organization of legumin genes in pea

We have compared physical and genetic maps of the region around the legJ gene in pea. In this vicinity there are four B-type legumin genes, arranged as two close pairs. The detection of a recombination event within this gene cluster allows the orientation of this group of genes within the surrounding linkage group to be determined. The relationship between physical and genetic distances in this region is discussed, as are the implications of this for relating physical and genetic maps elsewhere in the pea genome.

Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172

In Arabidopsis, the identity of perianth and reproductive organs are specified by antagonistic action of two floral homeotic genes, APETALA2 (AP2) and AGAMOUS (AG). AP2 is also negatively regulated by an evolutionary conserved interaction with a microRNA, miR172, and has additional roles in general plant development. A kiwifruit gene with high levels of homology to AP2 and AP2-like genes from other plant species was identified. The transcript was abundant in the kiwifruit flower, particularly petal, suggesting a role in floral organ identity. Splice variants were identified, all containing both AP2 domains, including a variant that potentially produces a shorter transcript without the miRNA172 targeting site. Increased AP2 transcript accumulation was detected in the aberrant flowers of the mutant 'Pukekohe dwarf' with multiple perianth whorls and extended petaloid features. In contrast to normal kiwifruit flowers, the aberrant flowers failed to accumulate miR172 in the developing whorls, although accumulation was detected at the base of the flower. An additional role during dormancy in kiwifruit was proposed based on AP2 transcript accumulation in axillary buds before and after budbreak.

Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.

Genome-wide SNP detection, validation, and development of an 8K SNP array for apple

As high-throughput genetic marker screening systems are essential for a range of genetics studies and plant breeding applications, the International RosBREED SNP Consortium (IRSC) has utilized the Illumina Infinium® II system to develop a medium- to high-throughput SNP screening tool for genome-wide evaluation of allelic variation in apple (Malus×domestica) breeding germplasm. For genome-wide SNP discovery, 27 apple cultivars were chosen to represent worldwide breeding germplasm and re-sequenced at low coverage with the Illumina Genome Analyzer II. Following alignment of these sequences to the whole genome sequence of 'Golden Delicious', SNPs were identified using SoapSNP. A total of 2,113,120 SNPs were detected, corresponding to one SNP to every 288 bp of the genome. The Illumina GoldenGate® assay was then used to validate a subset of 144 SNPs with a range of characteristics, using a set of 160 apple accessions. This validation assay enabled fine-tuning of the final subset of SNPs for the Illumina Infinium® II system. The set of stringent filtering criteria developed allowed choice of a set of SNPs that not only exhibited an even distribution across the apple genome and a range of minor allele frequencies to ensure utility across germplasm, but also were located in putative exonic regions to maximize genotyping success rate. A total of 7867 apple SNPs was established for the IRSC apple 8K SNP array v1, of which 5554 were polymorphic after evaluation in segregating families and a germplasm collection. This publicly available genomics resource will provide an unprecedented resolution of SNP haplotypes, which will enable marker-locus-trait association discovery, description of the genetic architecture of quantitative traits, investigation of genetic variation (neutral and functional), and genomic selection in apple.

Characterisation of a glutathione reductase gene and its genetic locus from pea (Pisum sativum L.)

A cDNA encoding the chloroplast/mitochondrial form of glutathione reductase (GR:EC 1,6,4,2) from pea (Pisum sativum L.) was used to map a single GR locus, named GORI. In two domesticated genotypes of pea (cv, Birte and JI 399) it is likely that the GORI locus contains a single gene. However, in a semi-domesticated land race of pea sequences were detected but closely related sets of GR gene sequences were in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from ev. Birte, sequenced and its structure analysed. No features of the transcription or structure of the gene suggested a mechanism for generating any more than one form of . From these data plus previously published biochemical evidence was suggested a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GORI-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was present which could perhaps account for the generation of multiple isoforms of GR. The mismatch between the number of charge-separable isoforms in pea and the proposed number suggests that different GR isoforms arise by some form of post-transnational modification.

The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Fruit softening in apple (Malus 3 domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A coldrelated gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the coldand ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 l of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.

Macroscopic fundamental diagram for Brisbane, Australia: empirical findings on network partitioning and incident detection

The Macroscopic Fundamental Diagram (MFD) relates space-mean density and flow, and the existence with dynamic features was confirmed in congested urban network in downtown Yokohama with real data set. Since the MFD represents the area-wide network traffic performances, studies on perimeter control strategies and an area traffic state estimation utilizing the MFD concept has been reported. However, limited works have been reported on real world example from signalised arterial network. This paper fuses data from multiple sources (Bluetooth, Loops and Signals) and presents a framework for the development of the MFD for Brisbane, Australia. Existence of the MFD in Brisbane arterial network is confirmed. Different MFDs (from whole network and several sub regions) are evaluated to discover the spatial partitioning for network performance representation. The findings confirmed the usefulness of appropriate network partitioning for traffic monitoring and incident detections. The discussion addressed future research directions.

The ranking based constrained document clustering method and its application to social event detection

With the growing size and variety of social media files on the web, it’s becoming critical to efficiently organize them into clusters for further processing. This paper presents a novel scalable constrained document clustering method that harnesses the power of search engines capable of dealing with large text data. Instead of calculating distance between the documents and all of the clusters’ centroids, a neighborhood of best cluster candidates is chosen using a document ranking scheme. To make the method faster and less memory dependable, the in-memory and in-database processing are combined in a semi-incremental manner. This method has been extensively tested in the social event detection application. Empirical analysis shows that the proposed method is efficient both in computation and memory usage while producing notable accuracy.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

Exploring the scope of gene patents through new levels of transparency

As the global intellectual property (IP) system grows and now impacts virtually all citizens, it is crucial that the means to understand these rights and their teachings, as well as their implications and scope become global public goods. To do so requires not only that the primary data is available freely and openly in a standardized and re-useable form, but that tools to visualize, analyse and model that data are similarly open and free public goods, adaptable to diverse needs and uses; this we call ‘transparency’.

Recursive actuator fault detection and diagnosis for emergency landing of UASs

This paper presents a practical recursive fault detection and diagnosis (FDD) scheme for online identification of actuator faults for unmanned aerial systems (UASs) based on the unscented Kalman filtering (UKF) method. The proposed FDD algorithm aims to monitor health status of actuators and provide indication of actuator faults with reliability, offering necessary information for the design of fault-tolerant flight control systems to compensate for side-effects and improve fail-safe capability when actuator faults occur. The fault detection is conducted by designing separate UKFs to detect aileron and elevator faults using a nonlinear six degree-of-freedom (DOF) UAS model. The fault diagnosis is achieved by isolating true faults by using the Bayesian Classifier (BC) method together with a decision criterion to avoid false alarms. High-fidelity simulations with and without measurement noise are conducted with practical constraints considered for typical actuator fault scenarios, and the proposed FDD exhibits consistent effectiveness in identifying occurrence of actuator faults, verifying its suitability for integration into the design of fault-tolerant flight control systems for emergency landing of UASs.