The application of metallointercalators in recognition of and charge transport in nucleic acids

Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety bind to DNA through the major groove. These metallointercalators can recognize DNA sites and perform reactions on DNA as a substrate. The site-specific metallointercalator Λ-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription factor, yAP-1, from an oligonucleotide that contains a common binding site. The demonstration that metal complexes can prevent transcription factor binding to DNA site-specifically is an important step in using metallointercalators as therapeutics.

The distinctive photochemistry of metallointercalators can also be applied to promote long range charge transport in DNA. Experiments using duplexes with regions 4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying order showed that radical migration is more dependent on the sequence of bases, and less dependent on the distance between the guanine doublets. This result suggests that mechanistic proposals of long range charge transport must involve all the bases.

RNA/DNA hybrids show charge migration to guanines from a remote site, thus demonstrating that nucleic acid stacking other than B-form can serve as a radical bridge. Double crossover DNA assemblies also provide a medium for charge transport at distances up to 100 Å from the site of radical introduction by a tethered metal complex. This radical migration was found to be robust to mismatches, and limited to individual, electronically distinct base stacks. In single DNA crossover assemblies, which have considerably greater flexibility, charge migration proceeds to both base stacks due to conformational isomers not present in the rigid and tightly annealed double crossovers.

Finally, a rapid, efficient, gel-based technique was developed to investigate thymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated via the bases of a thymine-thymine interface; reversal of this ligation is easily visualized by gel electrophoresis. This assay was used to show that the repair of thymine dimers from a distance through DNA charge transport can be accomplished with different photooxidants.

Thus, nucleic acids that support long range charge transport have been shown to include A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a method for thymine dimer repair detection using charge transport was developed. These observations underscore and extend the remarkable finding that DNA can serve a medium for charge transport via the heteroaromatic base stack.

Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.

These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.

On innate and specific aspects of human language

Mixel Aurnague, Kepa Korta and Jesus M. Larrazabal (eds.)

Tests of some specific effect substances in duck nutrition. Translation from: Biologisace a chemisaze vyzivy svirat 5, 43-48, 1969.]

There is a constantly increasing collection of manufactured substances, whose effectiveness in the sustenance of ducks is under investigation. The author examines the effects of some substances already previously tested and also there were examined substances which had not hitherto been studied. The use of different supplements for late autumn fattening is studied through various experiments.

Site-Specific Isotopes in Small Organic Molecules

Stable isotope geochemistry is a valuable toolkit for addressing a broad range of problems in the geosciences. Recent technical advances provide information that was previously unattainable or provide unprecedented precision and accuracy. Two such techniques are site-specific stable isotope mass spectrometry and clumped isotope thermometry. In this thesis, I use site-specific isotope and clumped isotope data to explore natural gas development and carbonate reaction kinetics. In the first chapter, I develop an equilibrium thermodynamics model to calculate equilibrium constants for isotope exchange reactions in small organic molecules. This equilibrium data provides a framework for interpreting the more complex data in the later chapters. In the second chapter, I demonstrate a method for measuring site-specific carbon isotopes in propane using high-resolution gas source mass spectrometry. This method relies on the characteristic fragments created during electron ionization, in which I measure the relative isotopic enrichment of separate parts of the molecule. My technique will be applied to a range of organic compounds in the future. For the third chapter, I use this technique to explore diffusion, mixing, and other natural processes in natural gas basins. As time progresses and the mixture matures, different components like kerogen and oil contribute to the propane in a natural gas sample. Each component imparts a distinct fingerprint on the site-specific isotope distribution within propane that I can observe to understand the source composition and maturation of the basin. Finally, in Chapter Four, I study the reaction kinetics of clumped isotopes in aragonite. Despite its frequent use as a clumped isotope thermometer, the aragonite blocking temperature is not known. Using laboratory heating experiments, I determine that the aragonite clumped isotope thermometer has a blocking temperature of 50-100°C. I compare this result to natural samples from the San Juan Islands that exhibit a maximum clumped isotope temperature that matches this blocking temperature. This thesis presents a framework for measuring site-specific carbon isotopes in organic molecules and new constraints on aragonite reaction kinetics. This study represents the foundation of a future generation of geochemical tools for the study of complex geologic systems.

Studies of dinucleoside monophosphates and monomer-polynucleotide interactions by proton magnetic resonance

The nature of the intra- and intermolecular base-stacking interactions involving several dinucleoside monophosphates in aqueous solution have been investigated by proton magnetic resonance spectrosocopy, and this method has been applied to a study of the interaction of polyuridylic acid with purine and adenosine monomers.

The pmr spectra of adenylyl (3' → 5') cytidine (ApC) and cytidylyl (3' → 5') adenosine (CpA) have been studied as a function of concentration and temperature. The results of these studies indicate that the intramolecular base-stacking interactions between the adenine and cytosine bases of these dinucleoside monophosphates are rather strong, and that the stacking tendencies are comparable for the two sequence isomers. The chemical shifts of the cytosine H₅ and adenine H₂ protons, and their variations with temperature, were shown to be consistent with stacked conformations in which both bases of the dinucleoside monophosphates are preferentially oriented in the anti conformation as in similar dApdC, and dCpdA (dA = deoxyadenosine; dC = deoxycytidine) segments in double helical DNA. The intramolecular stacking interaction was found to have a pronounced effect on the conformations of the ribose moieties, and these conformational changes are discussed. The concentration studies indicate extensive self-association of these dinucleoside monophosphates, and analysis of the concentration data facilitated determination of the dimerization constant for the association process as well as the nature of the intermolecular complexes.

The dependence of the ribose conformation upon the extent of intramolecular base-stacking was used to demonstrate that the base-base interaction in cytidylyl (3' → 5') cytidine (CpC) is rather strong, while there appears to be little interaction between the two uracil bases of uridylyl (3' → 5') uridine (UpU).

Studies of the binding of purine to several ribose and deoxyribose dinucleoside monophosphates show that the mode of interaction is base-stacking, and evidence for the formation of a purine-dinucleoside monophosphate intercalated complex is presented. The purine proton resonances are markedly broadened in this complex, and estimates of the purine linewidths in the complex and the equilibrium constant for purine intercalation are obtained.

A study of the interaction of unsubstitued purine with polyuridylic acid at 29°C by pmr indicated that purine binds to the uracil bases of the polymer by base-stacking. The severe broadening of the purine proton resonances observed provides strong evidence for the intercalation of purine between adjacent uracil bases of poly U. This interaction does not result in a more rigid or ordered structure for the polymer.

Investigation of the interaction between adenosine and polyuridylic acid revealed two modes of interaction between the monomer and the polymer, depending on the temperature. At temperatures above 26°C or so, monomeric adenosine binds to poly U by noncooperative A-U base stacking. Below this temperature, a rigid triple-stranded 1A:2U complex is formed, presumably via cooperative hydrogen-bonding as has previously been reported.

These results clearly illustrate the importance of base-stacking in non-specific interactions between bases, nucleosides and nucleotides, and also reveal the important role of the base-stacking interactions in cooperatively for med structures involving specific base-pairing where both types of interaction are possible.

Studies of bacteriophage T4 tail fibers and tail fiber genes

The distal half of the bacteriophage T4 tail fiber interacts with the surface of the bacterium during adsorption. The largest polypeptide in this half fiber is the product of gene 37 (P37). During assembly of the tail fiber, P37 interacts with the product of gene 38 (P38). These two gene products are incompatible with the corresponding gene products from the related phage T2. T2 P37 does not interact with T4 P38 and T2 P38 does not interact with T4 P37. Crosses between T2 and T4 phages mutant in genes 37 and 38 have shown that the carboxyl end of P37 interacts with P38 and with the bacterial surface. In the corresponding region of gene 37 and in gene 38 there is no recombination between T2 and T4. In the rest of gene 37 there are two small regions with relatively high recombination and a region of low recombination.

When T2/T4 heteroduplex DNA molecules are examined in the electron microscope four nonhomologous loops appear in the region of genes 37 and 38. Heteroduplexes between hybrid phages which have part of gene 37 from T4 and part from T2 have roughly located gene 37 mutations in the heteroduplex pattern. For a more precise location of the , mutations a physical map of gene 37 was constructed by determining the molecular weights of amber polypeptide fragments on polyacrylamide gels in the presence of sodium dodecyl sulfate. When the physical and heteroduplex maps are aligned, the regions of low recombination correspond to regions of nonhomology between T2 and T4. Regions with relatively high recombination are homologous.

The molecular weight of T2 P37 is about 13,000 greater than that of T4 P37. Analysis of hybrid phage has shown that this molecular weight difference is all at the carboxyl end of P37.

An antiserum has been prepared which is specific for the distal half fiber of T4. Tests of the ability of gene 37 hybrids to block this antiserum show that there are at least 4 subclasses of antigen specified by different parts of P37.

Observations in the electron microscope of the tailfiber - anti- body complexes formed by the gene 37 hybrids and the specific anti- serum have shown that P37 is oriented linearly in the distal half fiber with its N-terminus near the joint between the two half fibers and its C-terminus near the tip of the fiber. These observations lead to a simple model for the structure of the distal half fiber.

The high recombination in T4 gene 34 was also investigated. A comparison of genetic and physical maps of gene 34 showed that there is a gradient of increasing recombination near one end of the gene.

Coma measurement by use of an alternating phase-shifting mask mark with a specific phase width

The correlation between the coma sensitivity of the alternating phase-shifting mask (Alt-PSM) mark and the mark's structure is studied based on the Hopkins theory of partially coherent imaging and positive resist optical lithography (PROLITH) simulation. It is found that an optimized Alt-PSM mark with its phase width being two-thirds its pitch has a higher sensitivity to coma than Alt-PSM marks with the same pitch and the different phase widths. The pitch of the Alt-PSM mark is also optimized by PROLITH simulation, and the structure of p - 1.92 lambda/NA and pw = 2p/3 proves to be with the highest sensitivity. The optimized Alt-PSM mark is used as a measurement mark to retrieve coma aberration from the projection optics in lithographic tools. In comparison with an ordinary Alt-PSM mark with its phase width being a half its pitch, the measurement accuracies of Z(7) and Z(14) apparently increase. (C) 2009 Optical Society of America

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.