The major aim of the studies that this thesis is based on has been to better define the topological determinants of the formation of so-called helical hairpins during membrane protein assembly in the ER membrane. The helical hairpin is a basic folding unit in membrane proteins. It is composed of two closely spaced transmembrane helices with a short connecting loop and it is believed to be inserted into the membrane as one compact unit. It is becoming increasingly clear that the helical hairpin is a very common structural element in membrane proteins and a detailed understanding of its properties is of central importance. We demonstrate that the efficiency of formation of helical hairpins depends both on the overall length of the hydrophobic segment, on the amino acids flanking the transmembrane segment, and on the identity of the central, potentially turn-forming residues. We also show that interhelical hydrogen bonds between pairs of Asn or Asp residues can induce helical hairpin formation. A detailed topology mapping is also reported for the Escherichia coli inner membrane chloride channel YadQ, a protein for which the X-ray structure is known. Our results provide a critical test of the reporter fusion approach and offer new insights into the YadQ folding pathway. In summary, the results present in this thesis have increased our understanding of the determinants of membrane protein topology and structure. Furthermore, the information obtained can be used to improve current models for predictions of membrane protein topology.

Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, attI and attC resulting in excision and integration of short DNA elements called gene cassettes carrying an attC site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The attC of the aadA1-qacE cassette junction in Tn21 was characterized in detail. Like other attC sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of attC. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among attC sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of attC hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.

Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.

The synthesis of four homologous anthraquinones (AQ I-IV) bearing increasing lengths of polyethylene glycol (PEG) side chains and their binding to AT- and GC-rich DNA hairpins are reported. The molecules were designed such that the cationic charge is at a constant position and the ethylene glycol units chosen to allow significant increases in size with minimal changes in hydrophobicity. The mode and affinity of binding were assessed using circular dichroism (CD), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). The binding affinity decreased as the AQ chain length increased along the series with both AT- and GC-rich DNA. ITC measurements showed that the thermodynamic parameters of AQ I-IV binding to DNA exhibited significant enthalpy-entropy compensation. The enthalpy became more favorable while the entropy became less favorable. The correlation between enthalpy and entropy may involve not only the side chains, but also changes in the binding of water and associated counterions and hydrogen bonding. The interactions of AQ I-IV with GC-rich DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The kinetics of a homologous series of anthraquinone threading intercalators, AQT I-IV with calf thymus DNA was studied using the stopped-flow. The threading mechanisms of the anthraquinones binding to DNA showed sensitivity to their side chain length. Fitting of the kinetic data led to our proposal of a two step mechanism for binding of AQT I, bearing the shortest side chain, and a three step mechanism for binding of the three longer homologs. Binding involves formation of an externally bound anthraquinone-DNA complex, followed by intercalation of the anthraquinone for AQT I-IV, then isomerization to another complex with similar thermodynamic stability for AQT II-IV.

Embryos derived by somatic cell nuclear transfer (SCNT) produce few pregnancies that result in a live, healthy offspring. This has largely been attributed to the aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve the efficiency of cloning there is a great deal of research needed to determine the role of proteins involved in early embryonic reprogramming. In addition, studies are needed to determine effects on somatic and embryonic cell development as a result of altering these proteins. In this study we investigate the use of RNA interference in bovine somatic cells and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an enzyme responsible for maintenance methylation in mammalian cells. We designed our experiments to test whether or not knocking down the DNMT1 gene would lead to a decrease in global methylation. It is our hypothesis that using somatic cells with reduced methylation may be advantageous for increasing the efficiency of cloning via somatic cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral vector capable of delivering a gene that produces a short hairpin RNA targeting the mRNA of DNMT1. The construct included a sequence coding for green fluorescent protein (GFP) that will allow us to identify cells expressing the hairpin as well as a region coding for neomycin resistance so we could select for a pure population of transgenic cells to use for analysis. Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA suggests that our shRNAs are capable of an 80% knockdown. The protein blot of indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation as a result of the DNMT1 knockdown. However, double transduced cells with a high knockdown percentage of DNMT1 mRNA and protein became hypermethylated. The second experiment was conducted to determine the effect of injecting small interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic activation. This experiment was designed to give us information on the survivability and epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the sham injected oocytes. This treatment group also had decreased DNA methylation as determined by immunolabeling of methylated cytosine residues.

DNA double-strand breaks (DSBs) are lethal in eukaryotic cells if left unrepaired. In Saccharomyces cerevisiae the Mre11/Rad50/Xrs2 (MRX) complex is required for repair of DSBs through homologous recombination and nonhomologous end joining. Although Mre11 complexes exhibit 3'[rightwards arrow]5' exonuclease activity and endonuclease activity on DNA hairpin and single-stranded DNA overhang substrates in vitro, the role of the MRX complex in homologous recombination in vivo is not well understood. It has been shown to be specifically required for the processing of protein-conjugated DNA ends at DSBs during meiosis and hairpin-capped DSBs in mitotic cells and has been suggested that the Mre11 nuclease functions to remove damaged DNA ends. Recently, the Sae2 protein has been demonstrated to be involved in hairpin-capped DSBs and DNA end processing along with MRX in vivo. However, the Sae2 protein has no known homologs outside of fungi and no obvious motifs to suggest the function(s) of the Sae2 protein. We have purified recombinant Sae2 and MRX and report that the Sae2 protein itself is a single-stranded DNA endonuclease. The Sae2 protein stimulates the 3[rightwards arrow]5' exonuclease activity of the MRX complex. Also, the MRX complex can stimulate Sae2 nuclease activity to cleave ssDNA adjacent to DNA hairpin structures. The Sae2 protein also binds independently to double-stranded DNA and forms higher order protein-DNA complexes with MRX. These results provide biochemical evidence for functional cooperatively between MRX and Sae2 on DSBs and hairpin-capped DNA ends. / text

No description available.

Background: RNA has been recognized as a key player in cellular regulation in recent years. In many cases, non-coding RNAs exert their function by binding to other nucleic acids, as in the case of microRNAs and snoRNAs. The specificity of these interactions derives from the stability of inter-molecular base pairing. The accurate computational treatment of RNA-RNA binding therefore lies at the heart of target prediction algorithms. Methods: The standard dynamic programming algorithms for computing secondary structures of linear single-stranded RNA molecules are extended to the co-folding of two interacting RNAs. Results: We present a program, RNAcofold, that computes the hybridization energy and base pairing pattern of a pair of interacting RNA molecules. In contrast to earlier approaches, complex internal structures in both RNAs are fully taken into account. RNAcofold supports the calculation of the minimum energy structure and of a complete set of suboptimal structures in an energy band above the ground state. Furthermore, it provides an extension of McCaskill's partition function algorithm to compute base pairing probabilities, realistic interaction energies, and equilibrium concentrations of duplex structures.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18316号 / 農博第2041号 / 新制||農||1020(附属図書館) / 学位論文||H26||N4823(農学部図書室) / 31174 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 安達 修二, 教授 伏木 亨 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Yes / Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed.