The Tetrahymena thermophila rDNA minichromosome has been used as a model system for studying DNA replication. Previous studies have identified cis-acting replication determinants within the rDNA origin and promoter region including the type I element that is essential for replication initiation, fork progression and promoter activation. TIF1 is a non-ORC single strand-binding protein that binds the type I element in vivo. TIF1 binds opposing strands at the origin and promoter regions indicating that it may play a role in selectively marking these regions. In this dissertation, I use gene disruption to elucidate the role of TIF1 in replication. This work reveals that TIF1 represses rDNA origin firing, and is required for proper macronuclear S phase progression and division. Replication at the rDNA origin initiates precociously despite the observation that TIF1 mutants exhibit an elongated macronuclear S phase and a diminished rate of DNA replication. The amitotic macronucleus also displays delayed and abnormal division even though cells exit S phase with a wild-type macronuclear DNA content. Nuclear defects are also evident in the diploid micronucleus as TIF1 mutants contain fewer micronuclear chromosomes and are unable to pass genetic information to progeny. This defect is progressive as clonal mutant lines exhibit micronuclear instability during subsequent vegetative cell cycling. This work reveals that these macro- and micronuclear phenotypes may be the result of DNA damage as TIF1 mutants are hypersensitive to DNA damaging agents. This suggests that TIF1 mutants may have defects in the DNA damage response pathway. TIF1-deficient cells also incur DNA damage with no exogenous damaging agents. I propose that micro- and macronuclear defects witnessed in TIF1 mutant cells result from cells exiting S phase with compromised chromosomes due to the accumulation of DNA damage. Furthermore, TIF1 appears to play a role in the prevention, recognition or repair of DNA damage in addition to regulating rDNA replication and cell cycle progression and division. Additionally, TIF1 plays an essential role in the faithful propagation of both the macro- and micronuclear genomes.

Knowledge of phylogenetic relationships among taxa is essential for comparative and evolutionary genomic research. Here, we report reconstruction of the phylogenetic tree of the genus Gossypium containing cultivated cottons of importance in agriculture by using variation of nuclear repetitive DNA sequences. Genomic DNA was isolated from 87 available accessions of 35 species representing all eight basic genome groups of the genus Gossypium and analyzed to infer phylogeny of the genus and genome origin of its polyploid species. Twenty-two interspersed repeated sequence clones derived from G. hirsutum, each representing a repeated sequence family, were hybridized to the genomic DNA of the 35 species, respectively. Southern hybridization showed that 15 of the repetitive DNA sequences could be detected in all of the eight diploid genome groups, five were A genome-specific, and two were detected in some of the non D-genome groups. A total of 642 major restriction bands of repeated sequences were used for phylogenetic analysis of the species. A phylogenetic tree of the species was constructed, based on the parsimony method and evaluated by the bootstrap approach. The tree was consistent with those previously constructed with different methods in major clades in which the genealogical lineages of species are largely congruent with genome designations and geographical distribution; but significantly different branching among some of the species was observed. These results not only further confirm the previously phylogenetic analysis of the species and the utility of repetitive DNA sequences for phylogenetic analysis of the genus Gossypium, but also provide new insights into the phylogeny of the genus.

Maintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants.

Molecular dynamics simulations were conducted on a system consisting of a decamer DNA solvated by trehalose and water (molecular ratio= 1:2), to mimic a relatively dry state for the DNA molecule. Simulations were performed at two different temperatures, 300 K and 450 K. The B-form DNA structure was shown to be stable at both temperatures. The analysis of hydrogen bonds between trehalose/water and DNA revealed that trehalose and backbone DNA formed the largest number of hydrogen bonds and thus constituted the major effect of structural protection for DNA. The number of hydrogen bonds formed by each OH group of trehalose with the backbone DNA was compared. Different types of trehalose-DNA interactions were analyzed, with no prevalent pattern recognized. Diffusion constants for trehalose and water were also calculated, suggesting a glassy/viscose state of the simulation system. It is believed that trehalose protects DNA in the dry state through the network of hydrogen bonds built by the sugars, which reduces the structural fluctuations of DNA and prevents its denaturation.

Delicate epigenetic modifications are essential for production of spermatids during spermatogenesis. DNA methyltransferase 3 (DNMT3), the enzymes involved in adding a methyl group to unmodified DNA, contains three members: DNMT3A, DNMT3B and DNMT3L. The latter lacks methyltransferase activity, but was closely associated with spermatogenesis in many reports. According to the presentation of mature spermatids in testis, azoospermia could be separated into obstructive and non-obstructive categories. Non-obstructive azoospermia is spermatogenesis defective, germ cells absent in seminiferous tube is the most serious type. Therefore, we would like to find out if there are differential expression of DNMT3 family transcripts in testes of azoospermia patients from infertility clinic. Using RT-PCR and qPCR, we found only 5 (29.4%) expressed DNMT3L in 17 non-obstructive patients, whereas all 20 obstructive patients expressed. Both groups were similar in expression levels of DNMT3A and DNMT3B. Nuclei of spermatogonia and spermatocyte were the main immunohisto-chemical localization of DNMT3L protein. Lost of germ cells should be the cause of undetectable DNMT3L expression in azoospermia patients. By this founding, it could serve as an indicator for ability of male germ cell culture in further applications of assisted reproduction.

Differential scanning calorimetry (DSC), atomic force microscopy (AFM), 1H nuclear magnetic resonance (NMR), one-and two-dimensional fourier transform infrared spectroscopes (FTIR), size exclusion chromatography (SEC), dynamic light scattering (DLS), viscosity analyses have been used to investigate the miscibility behavior, specific interactions and supramolecular structures of poly(vinylbenzyl thymine-co-butyl methacrylate) (PVBT-co-PBMA, T-PBMA) blending with poly(vinylbenzyl adenine-co-styrene) (PVBA-co-PS, A-PS) through multiple hydrogen bonding complex upon varying the vinylbenzyl thymine and vinylbenzyl adenine contents in DNA-like copolymers. We describe FTIR and 1H NMR spectra reveal that hydrogen bonding between VBA and VBT exclusively. In addition, viscosity measurement, SEC, and DLS provide the formation of supramolecular network structure in this binary blend system. A miscibility window exists when the vinylbenzyl thymine and vinylbenzyl adenine fraction in the copolymer is greater than 11 mol% in the A-PS/T-PBMA blend system, as predicted using the Painter¡VColeman association model.

Characterization of the polyoma virus chromosome as a circular, double-stranded, supercoiled DNA (Weil and Vinograd, 1963; Vinograd et al.,-1965) made it apparent that the DNA enzymatic machinery faces a formidable problem in ensuring faithful replication of genetic material. These studies initiated a search for enzymatic activities that are capable of overcoming this topological barrier and led to the discovery of DNA topoisomerase I, originally denoted as omega protein (Wang, 1971), followed by DNA gyrase (Gellert et al., 1976) from Escherichia coli. It is now established that Escherichia coli encodes four distinct topoisornerases. These include topoisomerase I (topA) (Wang, 1971), topoisomerase II (gyrA and gyrB) (Gellert et al., 1976), topoisomerase III (topB) (DiGate and Marians, 1988) and topoisomerase IV (parC and parE) (Kato et al, 1990; 1992). These enzymes perform various non-overlapping functions in vivo (reviewed in Roca, 1995). Additionally, a 'reverse gyrase", capable of introducing positive supercoils into DNA, in an ATP dependent manner, has been characterized from hyperthermophiles (Kikuchi and Asai, 1984; Kozyavkin et al., 1994).

Deoxyribonucleic acid and ribonucleic acid under physiological condition are polyanions composed of heterocyclic bases linked through sugar phosphate backbone. Due to Watson-Crick base pairing, DNA exists in double-helical form between two antiparallel strands of nucleic acid. Different conformations of DNA is possible among which the B-DNA form is considered to be the most common, and it is a right-handed double-helix with base pairs stacked at the center. There are two well-defined grooves termed as major and minor grooves, each has characteristic width and depth. Most of the DNA binding proteins generally approach DNA through the major groove, while small molecules such as drugs, antitumor antibiotics,1 their synthetic analogue,2 carcinogens,3 and the transition metal complexes4 interact with DNA through minor groove. The nucleic acids function in the storage and transfer of genetic information. The function of cell expressions of proteins, synthesis of all bio-materials are directly or indirectly governed by the nucleic acid present in the body. Not only that, the origin of many diseases lie behind the structural modification or alterations in nucleic acids occur beyond our control.5 There are different drugs both natural and synthetic which are important in antibiotic chemotherapy, act against these diseases by interacting with DNA. Now to understand the actual mechanism of many diseases, how drugs interact with DNA and its specificity, binding sites of DNA, we need to develop molecules that modify or interact with biological molecules and such molecules can probe various structural aspects and type of interaction of macromolecular association complexes. One of such probe is the DNA cleaving agent. The potential scope of the utility of these compound is enormous and ranges from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents (Fe(BLM), calicheamicin) that may be effective against a variety of neoplastic diseases. They can also act as a structural probe (e.g. Fe(EDTA)2), drug / protein-DNA footprinting agent and affinity cleaving agent.

DNA-Netzwerke können als formgebende Muster (Template) zur Funktionalisierung technischer Oberflächen für die bottom-up Materialsynthese Verwendung finden. Zum Aufbau solcher Netzwerke werden synthetisch hergestellte DNA-Oligonukleotide hybridisiert. Im ersten Teil der Arbeit wird ein Weg beschrieben, wie die zum gezielten Aufbau einer vorgegebenen Struktur notwendigen DNA-Oligonukleotide hergeleitet werden können. Dies geschieht über eine schrittweise Zerlegung der Struktur in Bausteine, welche wiederum in die Oligonukleotide zerlegt werden. Weiterhin werden verschiedene DNA-Strukturen und Netzwerke hergestellt, welche mittels Gelelektrophorese, Atomkraft-, Elektronen- und Fluoreszensmikroskopie charakterisiert werden. Es wird gezeigt, wie die Herstellung von im Fluoreszensmikroskop beobachtbaren DNA-Verzweigungen möglich ist. Die Bildung von röhrenartigen Strukturen, welche sich mit der Zeit in zweidimensionale Netzwerke umlagern, wird anhand eines Modells zu Keimbildung und Keimwachstum erklärt. Für die gezielte Zusammenlagerung einer bestimmten Anzahl von Bausteinen werden mehrere Varianten untersucht. Bei den einzelnen Varianten sind große Unterschiede in der Steifigkeit feststellbar. Es werden Untersuchungen zum Einbau gebogener Abschnitte in DNA-Netzwerke durchgeführt. Dabei wird insbesondere auf Gestaltung der gebogenen Abschnitte zur Vermeidung von Fehlpaarungen eingegangen. Untersuchungen zur Metallisierung von Netzwerken zeigen, dass zur Beibehaltung der Struktur eine erhöhte Stabilität notwendig ist.

<p>The amount of information derived from sequencing the humangenome is leading to an exponential increase in the rate atwhich genes and genetic disorders are mapped and characterized.As a consequence, the demand for genetic testing is alsodramatically increasing. Screening and assaying methods, otherthan direct sequencing, are gradually becoming available,although with different robustness, sensitivity and throughput.In the work summarized in this thesis, attention was devotedprimarily to the improvement and the development of newtechniques for some of these methods.</p><p>Considering the role of capillary electrophoresis inmolecular diagnostics and an associated important phenomenon -electroosmotic flow (EOF) - a robust, reproducible procedurefor surface modification of the inner wall of capillaries wasreported (<i>Paper I</i>) and this method was beneficially employed insubsequent projects.</p><p>The effort to screen and characterize point mutations in theCACNA1A gene, responsible for the Familial Hemiplegic Migraine(FHM) disease, led to the optimization and validation of a verysensitive technique on slab gel called Double Gradient–Denaturing Gradient Gel Electrophoresis (DG-DGGE) (<i>Paper II</i>). A more reliable and robust method forSingle-Strand Conformation Polymorphism analysis (SSCP) bycapillary electrophoresis, making use of neutral pH buffers,was also developed (<i>Paper III</i>), while the next project resulted in thedevelopment of a high-throughput method for assaying knownpolymorphisms by multiplex solid-phase minisequencing inmulti-capillary format using a detection system based on liquidcore waveguiding (<i>Paper IV</i>).</p><p>As these and other methods, as well as most applications inmolecular diagnostics and molecular biology, depend on thepolymerase chain reaction (PCR), an effort was made to enhancethe throughput of this technology and to minimize reactionvolumes and costs. For this, the concept of a dynamic reactorwas employed, instead of static systems where the reactionmixture is exposed to temperature cycles in a confined space. Acontinuous flow of small-volume reaction mixtures, separated byan immiscible hydrophobic carrier fluid such as aperfluorocarbon, is transported in a hydrophobic tube throughthree zones, which are kept at constant temperatures optimizedfor denaturation, annealing and elongation (<i>Paper V</i>). If combined with a technique for automatedsample loading and collection (<i>Chapter 7</i>), this method should allow veryhigh-throughput miniaturized DNA amplification. A samplehandling concept using hydrophilic anchors is proposed, whichshould also be useful for other miniaturized reactions andchemical processing.</p><p>Finally, some possible alternative methods are discussed aswell as future trends.</p><p><b>Keywords:</b>Amplification, anchor, array, assay,capillary, DGGE, disease, disorder, DNA, droplet, dynamicreactor, electrophoresis, fluorescence, fluorocarbon, gel,genetic alteration, genomics, genotyping, high-throughput,hydrophobic, liquid lid, miniaturized reaction, minisequencing,molecular diagnostics, mutation, PCR, polymerase chainreaction, screening, segmented flow, single nucleotidepolymorphism, SNP, SSCP, test.</p>