Validation of an NSP-based (negative selection pattern) gene family identification strategy

<p>Abstract</p> <p>Background</p> <p>Gene family identification from ESTs can be a valuable resource for analysis of genome evolution but presents unique challenges in organisms for which the entire genome is not yet sequenced. We have developed a novel gene family identification method based on negative selection patterns (NSP) between family members to screen EST-generated contigs. This strategy was tested on five known gene families in Arabidopsis to see if individual paralogs could be identified with accuracy from EST data alone when compared to the actual gene sequences in this fully sequenced genome.</p> <p>Results</p> <p>The NSP method uniquely identified family members in all the gene families tested. Two members of the FtsH gene family, three members each of the PAL, RF1, and ribosomal L6 gene families, and four members of the CAD gene family were correctly identified. Additionally all ESTs from the representative contigs when checked against MapViewer data successfully identify the gene locus predicted.</p> <p>Conclusion</p> <p>We demonstrate the effectiveness of the NSP strategy in identifying specific gene family members in Arabidopsis using only EST data and we describe how this strategy can be used to identify many gene families in agronomically important crop species where they are as yet undiscovered.</p

Computational methods for the discovery and analysis of genes and other functional DNA sequences

The need for automating genome analysis is a result of the tremendous amount of genomic data. As of today, a high-throughput DNA sequencing machine can run millions of sequencing reactions in parallel, and it is becoming faster and cheaper to sequence the entire genome of an organism. Public databases containing genomic data are growing exponentially, and hence the rise in demand for intuitive automated methods of DNA analysis and subsequent gene identification. However, the complexity of gene organization makes automation a challenging task, and smart algorithm design and parallelization are necessary to perform accurate analyses in reasonable amounts of time. This work describes two such automated methods for the identification of novel genes within given DNA sequences. The first method utilizes negative selection patterns as an evolutionary rationale for the identification of additional members of a gene family. As input it requires a known protein coding gene in that family. The second method is a massively parallel data mining algorithm that searches a whole genome for inverted repeats (palindromic sequences) and identifies potential precursors of non-coding RNA genes. Both methods were validated successfully on the fully sequenced and well studied plant species, Arabidopsis thaliana --Abstract, page iv

A quantitative study of gene identification techniques based on evolutionary rationales

Current gene identification (GI) techniques typically rely on matching biological or chemical properties of specific genes, specific species, specific ecotypes, etc...In this thesis, a new automated GI technique is proposed, and compared against another computer-based technique proposed earlier. Both methods utilize EST data available from NCBI databases to discover previously unknown genes. The newly proposed method identifies one gene family at a time and is based on a distinctive negative selection pattern (NSP) of differences, which is seen between the coding regions of gene family members. The other technique, called ESTminer, attempts genome-wide gene family identification for any organism, by detecting single nucleotide polymorphisms between potential family members. In this thesis, a complete automated analysis of both techniques is presented --Abstract, page iii

Agronomic and quantitative trait loci analyses of yield and yield-related traits in pigeonpea.

Doctor of Philosophy in Plant Breeding. University of KwaZulu-Natal, Pietermaritzburg, 2017.Abstract available in PDF file

Genetic characterization of the Cy transposable element system at the Bz locus of Zea mays L.

The unstable bz-rcy allele arose by the insertion of a receptor element, rcy, into the Bz locus of a single gamete from the TEL population. Mutability of bz-rcy is controlled by the independently segregating regulatory element Cy (Cycler). In the absence of Cy, bz-rcy conditions a stable bronze aleurone. In the presence of Cy, bz-rcy conditions many small fully colored spots on a bronze background. These two elements, rcy and Cy, were previously undescribed;Genetic tests have established a relationship between Cy and the Mutator system. Cy is not functionally homologous to any of the non-Mutator transposable element systems;The number of genetically active Cy elements in a plant can increase or decrease via Cy transposition;Nonresponsive derivatives of bz-rcy have been isolated. A model has been established to explain the loss of the distal markers C and Sh coincident with the origin of some of these derivatives;The original isolate of bz-rcy often generates derivative alleles (states) that condition altered spotting patterns, e.g., reduced numbers of spots and/or larger and smaller spots, that reflect alterations in the frequency and timing of rcy excisions from bz-rcy. In contrast to receptors of the Ac and En(Spm) systems, which undergo changes of state only in the presence of the appropriate regulatory element, bz-rcy can undergo changes of state in the absence of an active Cy;Cy changes of state occur less frequently than those of bz-rcy. States of Cy have been isolated that induce reduced rates (but not altered timing) of rcy excision from bz-rcy. Many of the Cy states show progressive loss of function over succeeding generations;Many bz-rcy states and some Cy states have the ability to revert to fine-high spotting (cycling). Cycling is developmentally regulated and occurs only in the presence of Cy. A model has been proposed which evokes a form of reversible DNA modification to explain the cycling phenomenon and the origin of bz-rcy states in the absence of Cy;Of 47 diverse maize lines assayed, only two sources were found to contain strong Cy elements, i.e., Mutator-related stocks and the TEL population. Of the remaining lines, six, contained weak Cy elements and the rest lacked genetically detectable Cy elements

Are scrapie-susceptible sheep more productive?

A relationship between scrapie susceptibility, which is determined by PrP genotype, and valuable production traits has long been noted by sheep farmers, with many claiming that their 'best' sheep often are found to be susceptible to scrapie, or have close siblings that are susceptible to scrapie. There have been several historical anecdotal reports to support this observation, but only recently has the hypothesis, that scrapie-susceptible sheep are more productive, been investigated in scientific study. This thesis contains the results of several such studies and is concluded by an investigation into whether the current breeding strategies being encouraged in the UK would be effective at eliminating scrapie from the national sheep flock. In these studies, PrP genotype was compared to objective measures such as lamb weights and Estimating Breeding Values (EBVs), as well as to subjective measurements which were based on a farmer's judgement of their sheep, with varying results. Analysis of the subjective measurements, rating scores and culling records did not show any association with PrP genotype. The results of the analysis of the EBVs were variable, and inconsistent between farms, with susceptibility to scrapie being associated with both increased and decreased productivity. There was a small association between PrP genotype and lamb weights, which indicated that at eight weeks of age, ARR/ARR lambs were slightly smaller than lambs of other more susceptible genotypes. Overall, however, there is no strong evidence that scrapiesusceptible sheep truly are more productive. The final section has shown that with the current suggested breeding strategies, there will still be some risk of scrapie outbreaks in some flocks

Obesity-Related Metabolome and Gut Microbiota Profiles of Juvenile Göttingen Minipigs—Long-Term Intake of Fructose and Resistant Starch

publishedVersio

Tilstedeværelsen av en akutt fase-reaksjon hos lam med eksperimentell klassisk skrapesjuke indikerer et skifte mot en pro-inflammatorisk tilstand i det kliniske endestadiet

Classical scrapie in sheep is a transmissible and fatal neurodegenerative disease caused by the self-replicating and infectious prion protein, PrPSc, which is a conformational variant of the normal cellular prion protein, PrPC. The prion protein is a highly conserved glycoprotein encoded by the PRNP gene and therefore within the same host both PrPC and PRPSC have the same unique amino acid sequence and they only differ in their three-dimensional folded structure. Specific mutations at codons 136, 154 and 171 of the PRNP gene leads to single amino acid substitutions, and the most common polymorphisms give rise to five possible alleles and 15 PRNP genotypes found in sheep. The different alleles are highly associated with levels of susceptibility to classical scrapie, where A136R154R171 allele provides high genetic resistance and V136R154Q171 allele results in highly susceptible animals. On the basis of this association between PRNP genotype and susceptibility, many EU MSs have implemented national breeding for resistance programme with the aim of increasing distribution of ARR allele and reducing the distribution of VRQ allele. For almost 20 years, the EU TSE regulation has required surveillance within each country to establish prevalence of prion diseases and the different PRNP genotypes. Classical scrapie has a widespread distribution and incidence rate fluctuates due to the complex interaction between prion and host factors, and prevalence can only be estimated by ante mortem testing through active and passive surveillance. Transmission between sheep occurs through direct and indirect contact, and PrPSc can remain infective in the environment for years. The most common route of infection is the oral route, and infected animals can excrete PrPSc through foetal membranes and fluids, saliva, urine, faeces, and milk. Pathogenesis is highly influenced by PRNP genotype, as animals of the most susceptible genotypes have the most effective uptake of PrPSc across small intestine followed by an extensive dissemination and involvement of the SLOs, and an early neuroinvasion with spread of PrPSc within the CNS. The susceptible genotypes will contribute the most to spread of infectivity and environmental contamination. This work describes the results from experimental classical scrapie where homozygous VRQ lambs were inoculated orally at birth with homogenated brain material from either healthy sheep or from natural cases of classical scrapie. This resulted in a worst-case scenario type of classical scrapie with sudden onset of severe clinical signs at 22 wpi followed by a rapid deterioration and euthanasia at 23 wpi. Serum samples were collected at regular intervals and tissue samples from brain and liver were sampled at post mortem examination. Proteomic examinations of serum revealed a downregulation of several protein peaks during the pre-symptomatic incubation period in the scrapie affected group compared to the control group, and a shift to upregulation of protein peaks onwards from 22 wpi. Genomic examinations of serum samples showed a slight downregulation IL1B and TLR4 at 16 wpi, followed by a change at 22 wpi with upregulation of genes encoding TLRs, C3 and APPs. Genomic examination of liver and brain tissues showed an alteration in gene expression of APPs in accordance with an APR. Serum analyses of different APPs showed increased levels of the positive APPs and a reduced concentration of negative APPs. These findings are indicative of a shift from anti-inflammatory to pro-inflammatory systemic innate immune response that coincide with the onset of debilitating clinical disease. In neurodegenerative diseases, the innate immune response in the CNS has a key role in both onset and progression of disease and resolution of inflammation. The accumulation of PrPSc in the CNS has been associated with a chronic activation of the innate immune response, pro-inflammatory activation of microglia, neuroinflammation, and neurodegeneration. The diseases phenotype registered in this work is a result of PRNP genotype, and time and dose of inoculation, which can occur naturally if the right circumstances are in place. New-born homozygous VRQ lambs from an infected dam can get infected at birth. These cases could develop a similar disease progression as described in this work, resulting in an efficient and fast uptake and widespread peripheral and central dissemination of PrPSc, and clinical disease at a young age. These cases would present as a diagnostic challenge and easily missed as classical scrapie. Due to their young age, these cases would not be sampled through active surveillance. If incubation period extends commercial lifespan, these lambs would be slaughtered for human consumption, and due to their PRNP genotype, prions would enter the food chain. Control of classical scrapie can probably not be achieved by absence of infectivity, but absence of clinical disease is possible through breeding for resistance which will provide flock immunity to classical scrapie.Klassisk skrapesyke hos sau er en overførbar og dødelig nevrodegenerativ sykdom forårsaket av det selvrepliserende og smittsomme prionproteinet, PrPSc, som er en variant av det normale cellulære prionproteinet, PrPC. Prionproteinet er et glykoprotein som er kodet for av PRNP-genet. Dette betyr at PrPC og PRPSC hos samme verten, har den samme unike aminosyresekvensen og det er kun den tredimensjonale strukturen som skiller dem. Spesifikke mutasjoner ved kodonene 136, 154 og 171 i PRNP-genet fører til substitusjoner av enkelte aminosyrer, og de vanligste polymorfismer gir opphav til fem mulige alleler, og 15 PRNP-genotyper hos sau. De forskjellige allelene er assosiert med nivå av mottakelighet for klassisk skrapesyke, og A136R154R171-allel fører til genetisk resistens, og V136R154Q171-allel gir høy mottagelighet. På bakgrunn av denne sammenhengen mellom PRNP-genotype og mottakelighet, har mange EU medlemsland innført nasjonale avlsprogram som har mål om å øke utbredelsen av ARR-allel, og samtidig en reduksjon av VRQ-allel. I snart 20 år har EUs TSE-regelverk krevd nasjonale overvåkingsprogram for å bestemme forekomsten av prionsykdommer og kartlegge utbredelsen av de forskjellige PRNP-genotypene. Klassisk skrapesyke er utbredt, men forekomsten vil variere med bakgrunn i det komplekse samspillet mellom prionprotein og vertsfaktorer. Prevalens kan estimeres gjennom ante mortem testing i forbindelse med aktivt og passivt overvåkingsprogram. Smitteoverføring mellom sau skjer ved direkte og indirekte kontakt, og PrPSc er smittsomt i flere år i miljøet. Den vanligste infeksjonsveien er gjennom oralt inntak, og dyr kan skille ut smittsomt PrPSc via fosterhinner og væsker, spytt, urin, feces og melk, og nivå er styrt av PRNP genotype.Research Council of Norwa

Evaluation of three 3ABC ELISAs for foot-and-mouth disease non-structural antibodies using latent class analysis

BACKGROUND: Foot-and-mouth disease (FMD) is a highly contagious viral disease of even-toed ungulates. Serological diagnosis/surveillance of FMD presents several problems as there are seven serotypes worldwide and in the event of vaccination it may be necessary to be able to identify FMD infected/exposed animals irrespective of their vaccination status. The recent development of non-structural 3ABC protein (NSP) ELISA tests has greatly advanced sero-diagnosis/surveillance as these tests detect exposure to live virus for any of the seven serotypes of FMD, even in vaccinated populations. This paper analyses the performance of three NSP tests using a Bayesian formulation of the Hui-Walter latent class model to estimate test sensitivity and specificity in the absence of a "gold-standard" test, using sera from a well described cattle population in Cameroon with endemic FMD. RESULTS: The analysis found a high sensitivity and specificity for both the Danish C-ELISA and the World Organisation for Animal Health (O.I.E.) recommended South American I-ELISA. However, the commercial CHEKIT kit, though having high specificity, has very low sensitivity. The results of the study suggests that for NSP ELISAs, latent class models are a useful alternative to the traditional approach of evaluating diagnostic tests against a known "gold-standard" test as imperfections in the "gold-standard" may give biased test characteristics. CONCLUSION: This study demonstrates that when applied to naturally infected zebu cattle managed under extensive rangeland conditions, the FMD ELISAs may not give the same parameter estimates as those generated from experimental studies. The Bayesian approach allows for full posterior probabilities and capture of the uncertainty in the estimates. The implications of an imperfect specificity are important for the design and interpretation of sero-surveillance data and may result in excessive numbers of false positives in low prevalence situations unless a follow-up confirmatory test such as the enzyme linked immunoelectrotransfer blot (EITB) is used

Generation and Characterisation of Novel Monoclonal Antibodies towards Ovarian Tumour Stem Cells

Tumour stem cells (TSCs) are hypothesised to be a rare population of tumour cells which possess stem cell-like properties and are resistant to conventional therapy. Although cell surface markers have been widely used to characterise TSCs, previous literature suggests that no specific marker has been found for ovarian TSCs. We aimed to identify and characterise novel antibodies specific to ovarian epithelial TSCs, particularly towards populations with Hoechst efflux (Side Population, SP) and aldehyde dehydrogenase activity which are associated with a stem cell phenotype and drug resistance. Putative TSC subpopulations from ovarian tumour cell lines isolated by fluorescence activated cell sorting (FACS) using differential Hoechst dye uptake and Aldefluor activity assays displayed stem cell-like characteristics, including the upregulation of stem cell markers, increased anchorage-independent growth and increased invasive properties. A panel of monoclonal antibodies (mAbs) was then generated by injecting Aldefluor-positive ovarian tumour IGROV1 cells into female BALB/c mice. 34 antibodies were found to be specific to Aldefluor-positive cells and 2 of these enriched for SP cells. The 2 mAbs demonstrated cross-reactivity on human embryonic stem cells but no cross-reactivity to normal ovarian cell lines. Subpopulations of ovarian cell lines positive for the mAbs displayed stem cell-like characteristics, including upregulation of stem cell markers - CD133, ABCB1 and ALDH1A1. Sorted mAb populations were injected into non-obese diabetic/severe combined (NOD/SCID) mice and differential in vivo tumour formation was observed. Finally, the target antigen which both mAbs recognised was identified by mass spectrometry to be clathrin heavy chain (CHC1). We conclude that rare subpopulations with tumour-sustaining capability and stem cell-like characteristics can be identified in ovarian cancer using the 2 novel antibodies generated. Both mAbs target CHC1 on tumour-sustaining populations which are enriched for multiple stem cell markers and are therefore potential novel diagnostic markers and/or therapeutic agents