De Novo Transcriptome Sequence Assembly and Analysis of RNA Silencing Genes of Nicotiana benthamiana

Background: Nicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection. Methodology/Results: RNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription. Conclusions: The assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant. © 2013 Nakasugi et al.

Multiple analysis of three common genetic alterations associated with thrombophilia

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia. (C) 2000 Lippincott Williams and Wilkins.

Identification of early-stage colorectal cancer patients at risk of relapse post-resection by immunobead reverse transcription-PCR analysis of peritoneal lavage fluid for malignant cells

Purpose: Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse. Experimental Design: We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin γ2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse. Results: Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors. Conclusion: The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer. © 2006 American Association for Cancer Research.

A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding

Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover. © 2005 SGM.

Analysis of killer immunoglobulin-like receptor genes in ankylosing spondylitis

Objectives: To assess the possible association of killer immunoglobulin-like receptor (KIR) genes, specifically KIR3DL1, KIR3DS1 and KIR3DL2, with ankylosing spondylitis (AS). Methods: 14 KIR genes were genotyped in 200 UK patients with AS and 405 healthy controls using multiplex polymerase chain reaction. Sequence-specific oligonucleotide probes were used to subtype 368 cases with AS and 366 controls for 12 KIR3DL2 alleles. Differences in KIR genotypes and KIR3DL2 allele frequencies were assessed using the χp2p test. Results: KIR3DL1 and KIR3DS1 gene frequencies were very similar in cases with AS and controls (odds ratio = 1.5, 95% confidence interval 0.8 to 3.0, and odds ratio = 1.02, 95% confidence interval 0.2 to 5.3, respectively). KIR3DL2 allele frequencies were not significantly different between cases with AS and controls. Conclusions: Neither the KIR gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to AS.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent

Background The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. Results In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. Conclusions Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.

Entrepreneurial discovery and exploitation processes : sequence or symbiosis?

This study examined the effect that temporal order within the entrepreneurial discovery-exploitation process has on the outcomes of venture creation. Consistent with sequential theories of discovery-exploitation, the general flow of venture creation was found to be directed from discovery toward exploitation in a random sample of nascent ventures. However, venture creation attempts which specifically follow this sequence derive poor outcomes. Moreover, simultaneous discovery-exploitation was the most prevalent temporal order observed, and venture attempts that proceed in this manner more likely become operational. These findings suggest that venture creation is a multi-scale phenomenon that is at once directional in time, and simultaneously driven by symbiotically coupled discovery and exploitation.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci : a conservation alert?

Genetic variation at allozyme and mitochondrial DNA loci was investigated in the Australian lungfish, Neoceratodus forsteri Krefft 1870. Tissue samples for genetic analysis were taken non-lethally from 278 individuals representing two spatially distinct endemic populations (Mary and Burnett rivers), as well as one population thought to be derived from an anthropogenic translocation in the 1890's (Brisbane river). Two of 24 allozyme loci resolved from muscle tissue were polymorphic. Mitochondrial DNA nucleotide sequence diversity estimated across 2,235 base pairs in each of 40 individuals ranged between 0.000423 and 0.001470 per river. Low genetic variation at allozyme and mitochondrial loci could be attributed to population bottlenecks, possibly induced by Pleistocene aridity. Limited genetic differentiation was detected among rivers using nuclear and mitochondrial markers suggesting that admixture may have occurred between the endemic Mary and Burnett populations during periods of low sea level when the drainages may have converged before reaching the ocean. Genetic data was consistent with the explanation that lungfish were introduced to the Brisbane river from the Mary river. Further research using more variable genetic loci is needed before the conservation status of populations can be determined, particularly as anthropogenic demands on lungfish habitat are increasing. In the interim we recommend a management strategy aimed at conserving existing genetic variation within and between rivers.

A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa

Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91. 6 and 86. 6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV. © 2010 Springer-Verlag.

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

In arrayed ranks array technology in the study of mesothelioma steven

Mesothelioma is a rare malignancy arising from mesothelial cells lining the pleura and peritoneum. Advances in modern technology have allowed the development of array based approaches to the study of disease allowing researchers the opportunity to study many genes or proteins in a high-throughput fashion. This review describes the current knowledge surrounding array based approaches with respect to mesothelioma research. © 2009 by the International Association for the Study of Lung Cancer.