Sequencing by Synthesis (SBS), a DNA sequencing methodology based on the DNA polymerase reaction, is a promising paradigm for deciphering large-scale genomes. This thesis describes the design and synthesis of a variety of nucleotide reversible terminators (NRTs) with different characteristics. One set of NRTs possesses a phosphate moiety attached to the 2’ position of the sugar to block further incorporation in polymerase reaction, with the potential for fluorescent tag attachment at the same site or on the base through a cleavable linker for detection. The other set of NRTs possesses an azido-methyl moiety that blocks the 3’-hydroxyl group for detection by surface-enhanced Raman scattering. Each NRT has been tested in proof-of-principle SBS experiments. In addition, a set of 5’-phosphate tagged nucleotides has been developed and tested for nanopore electronic detection. A new set of NRTs, 2’-O-monophosphate 3’-hydroxyl nucleoside 5’-triphosphates (2’-P-NTPs) has been synthesized and its application for SBS has been investigated (chapter 2). These NRTs contain a phosphate at the 2’ position of the sugar ring, which serves as the removable capping group during the polymerase reaction. This moiety is positioned close to the 3’-hydroxyl group so as to block further nucleotide incorporation in the polymerase reaction. It nonetheless should allow improved binding to the polymerase relative to nucleotides with blocking groups at the 3’ position, since polymerases have strict requirements for the 3’-OH binding pocket. 2’-P-NTPs can be incorporated into the growing nucleic acid strand at temperatures ranging from 37oC to 65oC with Stoffel fragment modified 19 (SfM19) polymerase. After incorporation, the phosphate capping moiety on the 2’ position of the DNA extension product can be efficiently removed by enzymatic phosphatase reaction permitting the next incorporation step. Fluorescently labeled 2’-P-NTPs have the potential for sequencing DNA and direct sequencing of RNA-like templates. As an alternative to fluorescence-based SBS, a Raman spectroscopy detection method was developed using an azido moiety (N3) as both a 3’-OH blocking group and a label with an intense, narrow and unique Raman shift at 2125 cm-1, where virtually all biological molecules are transparent (chapter 3). First the four 3’-O-azidomethyl nucleotide reversible terminators (N3-dNTPs) were demonstrated to produce surface enhanced Raman scattering (SERS) at 2125 cm-1. These 4 nucleotide analogues were used as substrates for the polymerase to perform a complete 4-step SBS reaction. SERS was used to monitor the appearance of the azide-specific Raman peak at 2125 cm-1 as a result of polymerase mediated primer extension by a single N3-dNTP and disappearance of this Raman peak upon cleavage of the azido label to permit the next nucleotide incorporation, thereby determining the DNA sequence. Due to the small size of the azido label, the N3-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for this moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS. Chapter 4 describes the design, synthesis and characterization of a new set of 5’-phosphate labeled nano-tag nucleotides (NTNs) for single molecule electronic SBS by nanopore detection. Four modified oligonucleotide polymers that produce distinct electrical current blockade signals in nanopores were designed as the nano-tags. While most of the NTNs flow rapidly through the pore, those complementary to the nucleotide on the DNA template are captured by the polymerase and will have at least 10-fold longer dwell times in the pore, which affords enough time for measuring and discriminating the signals. Since the nano-tags are automatically removed during the polymerase extension reaction in real time, only natural DNA strands are produced. Thus this SBS method should decrease the overall sequencing time and increase the read length.

The first half of this dissertation demonstrates development of a novel method for DNA methylation profiling based on site specific conversion of cytosine in CpG sites catalyzed by DNA methyltransferases. DNA methylation, a chemical process by which DNA bases are modified by methyl groups, is one of the key epigenetic mechanisms used by cells to regulate gene expression. It predominantly occurs at the 5-position of cytosines in CpG sites and is essential in normal development. Aberrant methylation is associated with many diseases including cancer. Bisulfite Genomic Sequencing (BGS), the gold standard in DNA methylation profiling, works on the principle of converting unmethylated cytosines to uracils using sodium bisulfite under strong basic conditions that cause extensive DNA damage limiting its applications. This dissertation focuses on the research and development of a new method for single cell whole-genome DNA methylation profiling that will convert the unmethylated cytosines in CpG sites to thymine analogs with the aid of DNA methyltransferase and photo-irradiation. Previously we synthesized a model deoxycytidine containing an optimized allyl chemical group at the 5-position and demonstrated that this molecule undergoes photo-conversion to its deoxythymidine analog (C to T conversion) with irradiation at 300 nm. The C to T conversion also proved feasible using synthetic DNA molecules. In this thesis, we demonstrate the conversion of a novel modified deoxycytidine molecule (PhAll-dC) using 350 nm photo-irradiation and a triplet photosensitizer (thioxanthone, TX) to avoid potential DNA damage. The new photoproduct was identified as the deoxythymidine analog of the starting molecule as assessed by IR, MS and NMR. An AdoMet analog containing the optimized chemical group was also synthesized and tested for enzymatic transfer to the C5-position of CpG cytosines using DNA methyltransferases. DNA methyltansferase M.SssI was engineered for more efficient enzymatic transfer. In the future, we will incorporate a triplet photosensitizer into the photoreactive moiety on AdoMet to increase energy transfer efficiency for photo-conversion of C to the T analog. Incorporating this into an overall method followed by amplification and sequencing should allow us to assess the methylation status of all CpGs in the genome in an efficient manner. The second half of this dissertation demonstrates development of a DNA sequencing by synthesis (SBS) method, The Sequence Walking Approach, using novel nucleotide reversible terminators (NRTs) together with natural nucleotides. Following the completion of The Human Genome Project, next generation DNA sequencing technologies emerged to overcome the limitations of Sanger Sequencing, the prominent DNA sequencing technology of the time. These technologies led to significant improvements in throughput, accuracy and economics of DNA sequencing. Today, fluorescence-based sequencing by synthesis methods dominate the high-throughput sequencing market. One of the major challenges facing fluorescence-based SBS methods is their read length limitation which constitutes a big barrier for applications such as de novo genome assembly and resolving structurally complex regions of the genome. In this regard, we have developed a novel SBS method called ‘The Sequence Walking Approach’ to overcome current challenges in increasing the single pass read length of DNA sequencing. Our method utilizes three dNTPs together with one nucleotide reversible terminator in reactions called ‘walks’ that terminate at predetermined bases instead of after each incorporation. In this method, the primer extended via 4-color SBS is stripped off and replaced by the original primer for walking reactions. By reducing the accumulation of cleavage artifacts of incorporated NRTs in a single run, our method aims to reach longer read lengths. In this thesis, we have demonstrated a variation of The Sequence Walking Approach in which 4-color sequencing steps are interspersed with walking steps over a continuous length of DNA without stripping off extended primers and reannealing the original primer. The improvements introduced with this method will enable the use of fluorescence-based SBS in many applications such as detection of genomic variants and de novo genome assemblies while preserving low costs and high accuracy.

by Cheng Sau Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves [72]-[76]). / TABLE OF CONTENTS --- p.i / ABSTRACT --- p.iv / LIST OF FIGURES --- p.vi / LIST OF TABLES --- p.x / Chapter CHAPTER ONE --- RESEARCH BACKGROUND --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) --- p.2 / Chapter 1.2.1 --- Laser desorption methods --- p.2 / Chapter 1.2.2 --- The matrix --- p.3 / Chapter 1.2.2.1 --- Role of the matrix --- p.3 / Chapter 1.2.2.2 --- Features of the matrix --- p.4 / Chapter 1.2.3 --- Mechanisms of ion formation --- p.6 / Chapter 1.2.3.1 --- Desorption process(es) --- p.6 / Chapter 1.2.3.2 --- Ionization process(es) --- p.7 / Chapter 1.3 --- Sequencing of DNA --- p.8 / Chapter 1.3.1 --- DNA sequencing procedure --- p.10 / Chapter 1.3.1.1 --- Generation of the nested set of DNA molecules --- p.11 / Chapter 1.3.1.2 --- Sequence analysis --- p.11 / Chapter 1.3.2 --- MALDI-TOF-MS as a DNA sequencing tool --- p.12 / Chapter 1.3.3 --- MALDI analysis of oligonucleotides --- p.14 / Chapter 1.4 --- Outline of the present work --- p.16 / Chapter CHAPTER TWO --- INSTRUMENTATION AND EXPERIMENTAL --- p.18 / Chapter 2.1 --- Time-of-flight mass spectrometry (TOF-MS) --- p.18 / Chapter 2.1.1 --- Linear time-of-flight mass spectrometry --- p.18 / Chapter 2.1.2 --- Reflectron time-of-flight mass spectrometry --- p.21 / Chapter 2.1.3 --- Ion detection --- p.22 / Chapter 2.1.4 --- Vacuum system --- p.22 / Chapter 2.2 --- Instrumentation --- p.24 / Chapter 2.2.1 --- The laser system --- p.24 / Chapter 2.2.2 --- Ion source and vacuum system --- p.24 / Chapter 2.2.3 --- Flight tube and reflector --- p.27 / Chapter 2.2.4 --- The detector --- p.28 / Chapter 2.2.5 --- Data acquisition and computer control --- p.28 / Chapter 2.3 --- Experimental --- p.29 / Chapter 2.3.1 --- Sample preparation --- p.29 / Chapter 2.3.2 --- Mass spectrometric analysis --- p.30 / Chapter CHAPTER THREE --- USE OF AMMONIUM SALTS AS CO-MATRICES --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Experimental --- p.35 / Chapter 3.3 --- Results and Discussion --- p.36 / Chapter 3.3.1 --- Effects of counter-anions --- p.36 / Chapter 3.3.2 --- Effects of matrix materials --- p.40 / Chapter 3.4 --- Conclusions --- p.43 / Chapter CHAPTER FOUR --- USE OF POTASSIUM SALTS AS CO-MATRICES --- p.44 / Chapter 4.1 --- Introduction --- p.44 / Chapter 4.2 --- Experimental --- p.44 / Chapter 4.3 --- Results and Discussion --- p.44 / Chapter 4.3.1 --- Adduct formation --- p.49 / Chapter 4.3.2 --- Signal enhancement --- p.50 / Chapter 4.4 --- Conclusions --- p.52 / Chapter CHAPTER FIVE --- ANALYSIS OF HIGH MASS OLIGONUCLEOTIDES --- p.53 / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Experimental --- p.53 / Chapter 5.3 --- Results and Discussion --- p.54 / Chapter 5.4 --- Conclusions --- p.67 / Chapter CHAPTER SIX --- CONCLUSIONS AND FURTHER WORK --- p.68 / Chapter 6.1 --- Conclusions --- p.68 / Chapter 6.2 --- Further work --- p.70 / ACKNOWLEDGMENT --- p.A1 / REFERENCES --- p.R1 - R5

by Chu Kin Kan Astley. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xi-xx (3rd gp.)). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.iv / Table of Contents --- p.v / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Popularity of Shiitake Mushroom --- p.1 / Chapter 1.2 --- Inheritance of Genetic Materials in L.edodes --- p.1 / Chapter 1.3 --- Genetic Markers and Linkage Maps of L.edodes --- p.2 / Chapter 1.4 --- Aims of Study --- p.5 / Chapter Chapter 2 --- Mapping of Sequence Tagged Sites (STSs) and Expressed Sequence Tags (ESTs) on the Linkage Map of L.edodes by PCR-Single Strand Conformational Polymorphism (SSCP) Test / Chapter 2.1 --- Literature Review --- p.7 / Chapter 2.1.1 --- Construction of Genetic Linkage Map --- p.7 / Chapter 2.1.2 --- Logarithm of the Odds (LOD) Score --- p.8 / Chapter 2.1.3 --- MAPMAKER Program --- p.10 / Chapter 2.1.4 --- Sequence Tagged Site (STS) and Expressed Sequence Tag (EST) --- p.11 / Chapter 2.1.5 --- Polymerase Chain Reaction - Single Strand Conformational Polymorphism (PCR-SSCP) --- p.13 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1. --- Detection of the STS and EST markers with PCR-SSCP test --- p.18 / Chapter 2.2.1.1 --- Biological Material and Growth Conditions --- p.18 / Chapter 2.2.1.2 --- DNA Samples Preparation --- p.18 / Chapter 2.2.1.3 --- PCR Primers Designation --- p.20 / Chapter 2.2.1.4 --- PCR Amplification --- p.21 / Chapter 2.2.1.5 --- SSCP Test --- p.21 / Chapter 2.2.1.6 --- Silver Staining of the Polyacrylamide Gel --- p.22 / Chapter 2.2.2. --- Mapping of the STS and EST Markers --- p.22 / Chapter 2.2.2.1 --- Biological Material and Growth Conditions --- p.22 / Chapter 2.2.2.2 --- SSI DNA Preparation --- p.22 / Chapter 2.2.2.3 --- PCR-SSCP Test among SSIs --- p.24 / Chapter 2.2.2.4 --- Chi Square (X2) Test --- p.24 / Chapter 2.2.2.5 --- LOD Score Test and Mapping of the Markers --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Detection of the STS and EST Markers from PCR-SSCP Test --- p.27 / Chapter 2.3.1.1 --- DNA Sample Preparation --- p.27 / Chapter 2.3.1.2 --- Primers Designed for STS and EST Amplification --- p.27 / Chapter 2.3.1.3 --- Size Differences between Experimental and Expected PCR Products --- p.38 / Chapter 2.3.1.4 --- Markers of PCR Polymorphism (PCRP) --- p.38 / Chapter 2.3.1.5 --- Markers of PCR-SSCP and PCR Length Polymorphism (PCR-LP) --- p.42 / Chapter 2.3.2 --- Mapping of the STS/EST Markers --- p.49 / Chapter 2.3.2.1 --- DNA Templates of SSIs --- p.49 / Chapter 2.3.2.2 --- Polymorphism Profiles of STSs and ESTs among the L54-SSIs --- p.49 / Chapter 2.3.2.3 --- Chi-square Test --- p.55 / Chapter 2.3.2.4 --- Repeated EST Markers --- p.58 / Chapter 2.3.2.5 --- Anonymous Expressed Sequence Tag - EST31 --- p.58 / Chapter 2.3.2.6 --- Linkage Analysis and Mapping of Markers --- p.59 / Chapter 2.3.2.6.1 --- Linkage Relationships between the16 STS/EST Markers --- p.59 / Chapter 2.3.2.6.2 --- Mapping of the STS/EST Markers onto the RAPD Linkage Map --- p.62 / Chapter 2.4 --- Discussion --- p.69 / Chapter 2.4.1 --- DNA Template Preparation --- p.69 / Chapter 2.4.2 --- Size Difference Between Expected and Experimental PCR Product --- p.69 / Chapter 2.4.3 --- PCR Polymorphism (PCRP) --- p.70 / Chapter 2.4.4 --- PCR-LP --- p.70 / Chapter 2.4.5 --- PCR-SSCP --- p.71 / Chapter 2.4.5.1 --- Primer Designed for PCR-SSCP --- p.71 / Chapter 2.4.5.2 --- Markers Producing Efficiency of PCR-SSCP Test --- p.72 / Chapter 2.4.6 --- Linkage Map of L.edodes --- p.73 / Chapter 2.4.6.1 --- Map Distance --- p.73 / Chapter 2.4.6.2 --- Linkage Groups --- p.74 / Chapter 2.4.6.3 --- Map Markers --- p.75 / Chapter Chapter 3 --- Mapping of Agronomic Features of L.edodes / Chapter 3.1 --- Literature Review --- p.77 / Chapter 3.1.1 --- Aroma Feature of L.edodes --- p.77 / Chapter 3.1.1.1 --- Volatile Compounds in Shiitake (L.edodes) Mushroom --- p.77 / Chapter 3.1.1.2 --- Fragrance Signature of Shiitake Mycelium --- p.79 / Chapter 3.1.2 --- Mapping of Quantitative Trait Loci (QTL) --- p.84 / Chapter 3.1.2.1 --- Complex Traits --- p.84 / Chapter 3.1.2.2 --- Quantitative Traits Locus (QTL) --- p.85 / Chapter 3.1.2.3 --- Maximum-likelihood Estimate in QTL mapping --- p.86 / Chapter 3.1.2.4 --- MAPMAKER/QTL --- p.87 / Chapter 3.2 --- Material and Methods --- p.88 / Chapter 3.2.1 --- Aroma feature of Mycelium --- p.88 / Chapter 3.2.1.1 --- Preliminary Screening of Volatiles in the SSI Mycelia of L.edodes --- p.88 / Chapter 3.2.1.1.1 --- Biological Material and Growth Conditions --- p.88 / Chapter 3.2.1.1.2 --- Volatile Extraction from SSI Mycelia --- p.88 / Chapter 3.2.1.1.3 --- Screening of Volatile Compounds with GC-MS --- p.89 / Chapter 3.2.1.2 --- Quantification of the Target Aromatic Volatile in the Mycelia of SSI and Parents --- p.90 / Chapter 3.2.1.2.1 --- Sample Preparations --- p.90 / Chapter 3.2.1.2.2 --- Quantification of the Target Volatile --- p.90 / Chapter 3.2.2 --- Measurement of Mycelial Growth --- p.91 / Chapter 3.2.3 --- Observation of Pigment Secretion during Mycelial Growth --- p.91 / Chapter 3.2.4 --- Locating Putative QTL on the Genetic Map of L.edodes --- p.92 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Aroma Feature --- p.93 / Chapter 3.3.1.1 --- Preliminary Screening of Volatiles in Mycelia of L.edodes --- p.93 / Chapter 3.3.1.2 --- Quantification of l-octen-3-ol in SSI Mycelia --- p.103 / Chapter 3.3.1.2.1 --- Sample Preparation --- p.103 / Chapter 3.3.1.2.2 --- l-Octen-3-ol contents in SSI Mycelia --- p.103 / Chapter 3.3.1.3 --- Mapping of QTL for l-octen-3-ol level on the genetic map --- p.106 / Chapter 3.3.2 --- Mycelial Growth Rate (MGR) --- p.111 / Chapter 3.3.2.1 --- Measurement of Mycelial Growth Rate --- p.111 / Chapter 3.3.2.2 --- Mapping of QTL for MGR on the genetic map --- p.111 / Chapter 3.3.3 --- Pigment Secretion form SSI mycelia --- p.116 / Chapter 3.4 --- Discussion --- p.118 / Chapter 3.4.1 --- Significance of the QTLs --- p.118 / Chapter 3.4.2 --- QTL for Aroma Feature --- p.119 / Chapter 3.4.2.1 --- Trait of Aroma: l-octen-3-ol level --- p.119 / Chapter 3.4.2.2 --- QTL of l-octen-3-ol level --- p.120 / Chapter 3.4.3 --- Mycelial Growth Rate (MGR) --- p.123 / Chapter 3.4.4 --- Pigment Secretion --- p.125 / Chapter Chapter 4 --- General Discussion and Conclusions --- p.127 / Chapter 4.1 --- Future Works --- p.127 / Chapter 4.1.1 --- Mapping of L.edodes Genes --- p.127 / Chapter 4.1.2 --- Characterizing and Mapping of Agronomic Traits --- p.128 / Chapter 4.2 --- Conclusions --- p.128 / Referencesxi

Orientador: Renate-Krause Sakate / Banca: Marcelo Agenor Pavan / Banca: Kelly Cristina Gonçalves Rocha / Banca: Antonio Carlos Maringoni / Banca: Daiana Bampi / Resumo: A alface pode ser infectada por diferentes tipos de vírus. O Lettuce mosaic virus- LMV foi por muitos anos considerado um dos mais frequentes e amplamente distribuídos mundialmente, podendo ocorrer em infecções simples e/ou mistas com Lettuce mottle virus, LeMoV, um provável sequivirus. A falta de informações a respeito dos aspectos biológicos e moleculares relacionados à classificação taxonômica e à transmissão do LeMoV, motivou a testar sua transmissão com afídeos e por sementes, avançar no sequenciamento do genoma viral e avaliar a incidência do vírus em importantes áreas produtoras de alface no estado de São Paulo. Em 2014, dentre um total de 118 plantas sintomáticas analisadas, 63 (53%) foram positivas para a presença de tospovírus, 11 (9%) foram positivas para LMV e, apenas 6 amostras (5%) foram positivas para LeMoV. Em 2015, 40 plantas (80%) estavam infectadas com tospovírus, e o LMV e LeMoV não foram detectados, indicando que o LeMoV não está limitando a produção de alface, pelo menos não durante o ano de 2014 e 2015. A transmissão desse vírus por sementes não foi verificada nas 832 sementes provenientes de plantas de alface infectadas com LeMoV, o que indica que este vírus provavelmente não seja transmitido por semente. Myzus persicae e Aphis gossypii não foram capazes de transmitir o LeMoV de uma planta de alface para outra. Quase toda a sequencia completa do genoma do LeMoV foi obtida e a presença de domínios conservados verificados na região da capa proteica (CP),... (Resumo completo, clicar acesso eletrônico abaixo) / eng / Doutor

The human c-myc gene is a cellular proto-oncogene composed of three exons and two introns. Transcription of c-myc is controlled by two promoters, Pl and P2. The activity of these promoters is regulated by many factors, such as cellular transcription factors E2F, YYl, and HSV-1 immediate-early proteins, ICPO, ICP4. Many regulatory elements located both upstream of and between P 1 and P2 have been identified, and some of these are required for optimum expression of c-myc. In this thesis research, a region downstream from P2 in the c-myc exon 1 was identified by its response to transactivation by HSV-1 immediate-early proteins, ICPO and ICP4. The purpose of this research was to examine this region for regulatory sites that respond to HSV-1 infection. I hypothesized that after HSV-1 infection, ICPO and ICP4 activate c-myc expression, in part, through regulatory sequences present in exon 1. To test for this hypothesis, reporter plasmids containing (I) the c-myc promoter (from - 101 bp relative to Pl) and exon 1 coupled to the bacterial CAT gene were constructed. (ii) The c-myc exon sequences used were either intact (wild-type) or they were constructed with various deletions. The activities of these plasmids were examined in transient expression assays. To analyze protein binding, electrophoretic mobility shift assay (EMSA) and completion EMSAs were carried out. The results from these experiments lead to the following conclusions: (i) ICP4 and ICPO serve as activators, whereas ICP27 inhibits c-myc gene expression. (ii) The region from +332 to +513 within the c-myc exon 1 contains an important element required for transactivation of the c-myc gene by HSV-1 proteins. (iii) Cellular proteins, including factor YYl, bind to the region from +332 to +513 in the c-myc exon 1. Although the exact mechanism by which HSV-1 immediate-early proteins regulate cmyc gene expression is still not clear, it gives rise to a possibility that this regulation is caused by turning on or activation of the cellular regulatory proteins by ICP4 and ICPO. The cellular proteins in turn activate the c-myc gene expression by interacting with the ciselement downstream from P2.

A regulatory element involved in the transcriptional activation of the major capsid protein (VP5) of herpes simplex virus type 1 was identified and characterized in this research project. Gel mobility shift assay with nuclear extracts from both uninfected and HSV-1 infected HeLa cells identified two major protein-DNA complexes involving the VP5 promoter. No viral specific complex found. DNase I and orthophenanthroline-cu+ footprint analyses in the same laboratory revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, mutated VP5 promoters with deletion and insertion around LBS were constructed and linked to a reporter gene, bacterial chloramphenicol acetyltransferase gene. The effect of mutations were tested in transient expression assay. Deletion of LBS resulted in seven to eight-fold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV immediate-early genes. These results indicated LBS is involved in the maximal transactivation of the VPS gene. A search of published gene sequences found the homologs of LBS exist in a number of HSV-1 By promoters, and other viral promoters, as well as cellar promoters. Some of these homologs have found involved in the transcription regulation.

Bibliography: leaves [127-137]

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Calvin L. Keeler, Dept. of Animal & Food Sciences. Includes bibliographical references.

We present research on the design, development and application of algorithms for DNA sequence analysis, with a focus on environmental DNA (metagenomes). We present an overview and primer on algorithm development for bioinformatics of metagenomes; work on frameshift detection in DNA sequencing data; work on a computational pipeline for the assembly, feature prediction, annotation and analysis of bacterial genomes; work on unsupervised phylogenetic clustering of metagenomic fragments using Markov Chain Monte Carlo methods; and work on estimation of bacterial genome plasticity and diversity, potential improvements to the measures of core and pan-genomes.