Bibliography: leaves 104-130. The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed.

Graduation date: 1993

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 80-87).

My research focuses on structures of the genomes of single-stranded RNA viruses. The first project is concerned with the sequence and secondary structure of HIV-1 RNA. Based on the secondary structure that Watts et al. determined, I performed a series of analysis and the results suggested that the abundance of Adenosines at the wobble position of the codons leads to an unusual structure with numerous unpaired nucleotides. The findings indicated how the virus balances evolutionary pressures on the genomic RNA secondary structure against pressures on the sequence of the viral proteins. The second project is the modeling of satellite tobacco mosaic virus (STMV). STMV is a T=1 icosahedral virus with a single piece of RNA that has 1058 nucleotides. X-ray crystallography studies of this RNA have revealed a structure containing 30 helices. The linkers between the helices, the possible structures at the interior of the icosahedron, and the sequence of the RNA were all missing in the crystal structure. To explore how the genome is organized within the protein capsid, I built a 3D model based on the RNA secondary structure predicted by Susan Schroeder. Being the first all-atom model of any virus, this model is highly correlated with the crystal structure; and the comparison with the in vitro structure of the same RNA supports the hypothesis that capsid protein plays an important role in RNA folding during assembly. The third project includes the modeling of bacteriophage MS2 (MS2) and the examination of the compactness of RNA in different viruses. MS2 is a T=3 RNA virus, and the cryo-EM studies have revealed a double-shell conformation of the genome. My final model of MS2 recaptures the double-shell structure of the RNA presented in the cryo-EM density. In addition, the predicted secondary structure that I used for the construction of the model shares a strong similarity with the in vitro structure determined in 1980s. This similarity contrasts with the striking difference between in vivo and in vitro RNA structures observed in STMV. Inspired by this finding, I examined the compactness of the RNA of several different viruses. The results strongly suggest that the RNAs of viruses requiring packaging signals have evolved to be structurally compact, which facilitates post-replicational RNA packaging. In contrast, viruses that do not depend on packaging signals probably adopt co-replicational RNA packaging.

Theoretical studies focusing on the nature of landscapes that correlate molecular sequences to molecular function have mainly been carried out in silico due to the vast amounts of data that are needed for analysis. In vitro selections of aptamers are a good model system to study theoretical questions at a experimental level. With the introduction of robotic platforms that conduct in vitro selections, it is now capable of producing significant amounts of data in a short time, making theoretical modeling with real experimental data attainable. I will be using a Biomek 2000 Laboratory Automation Workstation to carry out multiple in vitro nucleic acid selections in parallel. I will explore the sequence space to examine whether existing in vitro selection systems are optimal at isolating the best winning species. New methods will be introduced that will allow for the selection of identical targets with identical pools free of cross contamination on the open robotic system. This will open the doors to further conduct selections against other identical or highly similar targets, such as complex cellular targets. Finally, I will investigate the methods to improve the effectiveness at isolating aptamers against the highly complex lung cancer cell lines. These targets are highly challenging for isolating specific aptamers because of the great diversity of biomarkers found among them. Moreover, their highly morphological similarity of the cultured cells makes selections for specific aptamers very difficult. I explore the different methods that will allow for the generation of aptamers that can distinguish between non-small cell lung cancer and small cell lung cancer, and between non-small cell lung cancer and normal lung cells. Fine-tuning of this process is essential at transferring this process to automated platforms for large-scale generation of biosensors against tumor biomarkers.

The widespread occurrence among eukaryotes of sex and of mobile DNA sequences requires an evolutionary explanation, since both appear to reduce individual fitness. Both phenomena have been hypothesized to provide fitness advantages to populations, but such explanations require rather than explain the initial establishment of mobile elements and genes for sex. Genes encoding sexuality may invade asexual populations as molecular parasites, whose success then allows mobile elements to spread as parasites of sexual genomes. The prediction that mobile elements can invade only sexual populations was tested using isogenic sexual and asexual populations of Saccharomyces cerevisiae and the retrotransposon Ty3. Active Ty3 elements more consistently invaded sexual than asexual populations. In subsequent experiments involving selection on media containing ethanol as a carbon source or $ beta$-glycerophosphate as a limiting phosphorus source, transposition by galactose-induced Ty3 elements produced none of the mutations involved in adaptation to these media, and conferred no adaptive advantage among competing populations. The mean copy numbers of two mobile elements were unchanged by long-term sexual or asexual propagation of Chlamydomonas reinhardtii populations, because transposition by these elements occurred very rarely or had no effect on fitness. Sexual and asexual S. cerevisiae populations did not differ in their adaptation to galactose media, but sexual populations maintained on glucose had higher growth rates on both media than did asexual populations maintained on glucose, implying that selection against deleterious mutations was more effective in sexual populations.

Gene finding in eukaryotic genomes is an essential part of a comprehensive approach to modern systems biology. Most methods developed in the past rely on a combination of computational prediction and external information about gene structures from transcript sequences and comparative genomics. In the past, external sequence information consisted of a combination of full-length cDNA and expressed sequence tag (EST) sequences. Much improvement in prediction of genes and gene isoforms is promised by availability of RNA-seq data. However, productive use of RNA-seq for gene prediction has been difficult due to challenges associated with mapping RNA-seq reads which span splice junctions to prevalent splicing noise in the cell. This work addresses this difficulty with the development of methods and implementation of two new pipelines: 1/ a novel pipeline for accurate mapping of RNA-seq reads to compact genomes and 2/ a pipeline for prediction of genes using the RNA-seq spliced alignments in eukaryotic genomes. Machine learning methods are employed in order to overcome errors associated with the process of mapping short RNA-seq reads across introns and using them for determining sequence model parameters for gene prediction. In addition to the development of these new methods, genome annotation work was performed on several plant genome projects.

Nucleic Acid Sequence Based Amplification (NASBA) is a powerful in vitro technique for amplification. NASBA is routinely used in many fields of microbiology, including food microbiology, and most recently in the identification of forensic body secretions (saliva, tears, sweat, semen, vaginal secretions). NASBA has many advantages over the traditional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) including speed, high sample throughput and increased sensitivity. Proper selection of the sequence of importance and the designe of NASBA primers precisely for that sequence are the two most critical steps for any NASBA assay. Proper designe of NASBA primers includes important considerations such as product (amplicon) length, the addition of a T7 RNA polymerase promoter sequence at the 5’end of one primer, and a 3’AT rich region. Primer designing is, therefore, laborious and error-prone. Currently, no such software is available that facilitates primer designing for NASBA. In this study Java-based software for designing NASBA primers was developed which will enable rapid and specific NASBA primer designing for gene expression studies. The designed program focused on scripting minimum Java coding lines to reduce the time and storage space. Two codes were scripted for this software, a pseudo-code (for Java Program Developers) and Byte-code (for Windows operating system). Our results showed that Java is an efficient tool in searching sequences of interest within a gene (or mRNA), allowing for NASBA primers to be designed more quickly and effortlessly. The program has maximum memory storage capacity and allows the users to retrieve old data with reference to date or time. To test the practicality of the newly developed program, gene sequences of salivary mucin and amylase were examined to facilitate extraction of novel RNA tandem repeat element NASBA markers for human saliva forensic identification. Tandem repeats of non-coding portion with in the of human genome are highly polymorphic and considered best for forensic use. Currently, only 13 Short Tandem Repeat (STR) markers are available for forensic case work, which are not enough to establish a definitive link between the victim and suspects. Identification and validation of a new human body fluid tandem repeat markers is cruicial for achieving high through put results and to exonerate the innocent. / Access to thesis permanently restricted to Ball State community only / Department of Biology

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Biology

Bibliography: leaves 104-130. / vi, 130, [25] leaves, [13] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997