No abstract

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20-30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from approximately 7 x 10(7) M-1 for oligo(dT)10 to approximately 1.5 x 10(10) M-1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (omega) for RPA binding was estimated to be approximately 15.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70,32, and 14 kDa Characterization of the mechanism of chromosomal DNA replication is essential for understanding the process of cell growth. Eukaryotic chromosomes are very large and complex; therefore, the study of model systems has been vital to reach our current understanding of DNA replication. One such model system, the papovavirus simian virus 40 (SV40), has properties very similar to those of cellular chromosomes (see recent reviews in references 7, 27, and 46). The SV40 genome exists in the cell in an authentic chromatin structure. Replication of SV40 DNA is dependent upon cellular replication proteins and a single virally encoded protein, large T antigen. SV40 DNA replication initiates at a specific DNA sequence and then proceeds bidirectionally, as does chromosomal replication. The development of a cell-free replication reaction capable of replicating SV40 DNA (34) led to the identification and purification of seven cellular proteins required for catalyzing DNA synthesis (36, 47, 52). Studies from several laboratories have led to a basic understanding of the mechanism of SV40 DNA replication (36, 47, 52). Initiation of replication occurs at a well-defined sequence, the SV40 origin of replication (Fig. 1)

The many types of insect ear share a common sensory element, the chordotonal organ, in which sound-induced antennal or tympanal vibrations are transmitted to ciliated sensory neurons and transduced to receptor potentials. However, the molecular identity of the transducing ion channels in chordotonal neurons, or in any auditory system, is still unknown. Drosophila that are mutant for NOMPC, a transient receptor potential (TRP) superfamily ion channel, lack receptor potentials and currents in tactile bristles but retain most of the antennal sound-evoked response, suggesting that a different channel is the primary transducer in chordotonal organs. Here we describe the Drosophila Nanchung (Nan) protein, an ion channel subunit similar to vanilloid-receptor-related (TRPV) channels of the TRP superfamily. Nan mediates hypo-osmotically activated calcium influx and cation currents in cultured cells. It is expressed in vivo exclusively in chordotonal neurons and is localized to their sensory cilia. Antennal sound-evoked potentials are completely absent in mutants lacking Nan, showing that it is an essential component of the chordotonal mechanotransducer.

No abstract

The Na superionic conductor (aka Nasicon, NaZrSiPO, where 0 ≤ x ≤ 3) is one of the promising solid electrolyte materials used in advanced molten Na-based secondary batteries that typically operate at high temperature (over ∼270 °C). Nasicon provides a 3D diffusion network allowing the transport of the active Na-ion species (i.e., ionic conductor) while blocking the conduction of electrons (i.e., electronic insulator) between the anode and cathode compartments of cells. In this work, the standard Nasicon (NaZrSiPO, bare sample) and 10 at% Na-excess Nasicon (NaZrSiPO, Na-excess sample) solid electrolytes were synthesized using a solid-state sintering technique to elucidate the Na diffusion mechanism (i.e., grain diffusion or grain boundary diffusion) and the impacts of adding excess Na at relatively low and high temperatures. The structural, thermal, and ionic transport characterizations were conducted using various experimental tools including X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). In addition, an ab initio atomistic modeling study was carried out to computationally examine the detailed microstructures of Nasicon materials, as well as to support the experimental observations. Through this combination work comprising experimental and computational investigations, we show that the predominant mechanisms of Na-ion transport in the Nasicon structure are the grain boundary and the grain diffusion at low and high temperatures, respectively. Also, it was found that adding 10 at% excess Na could give rise to a substantial increase in the total conductivity (e.g., ∼1.2 × 10 S/cm at 300 °C) of Nasicon electrolytes resulting from the enlargement of the bottleneck areas in the Na diffusion channels of polycrystalline grains.

The ability to detect variations in humidity is critical for many animals. Birds, reptiles and insects all show preferences for specific humidities that influence their mating, reproduction and geographic distribution. Because of their large surface area to volume ratio, insects are particularly sensitive to humidity, and its detection can influence their survival. Two types of hygroreceptors exist in insects: one responds to an increase (moist receptor) and the other to a reduction (dry receptor) in humidity. Although previous data indicated that mechanosensation might contribute to hygrosensation, the cellular basis of hygrosensation and the genes involved in detecting humidity remain unknown. To understand better the molecular bases of humidity sensing, we investigated several genes encoding channels associated with mechanosensation, thermosensing or water transport. Here we identify two Drosophila melanogaster transient receptor potential channels needed for sensing humidity: CG31284, named by us water witch (wtrw), which is required to detect moist air, and nanchung (nan), which is involved in detecting dry air. Neurons associated with specialized sensory hairs in the third segment of the antenna express these channels, and neurons expressing wtrw and nan project to central nervous system regions associated with mechanosensation. Construction of the hygrosensing system with opposing receptors may allow an organism to very sensitively detect changes in environmental humidity.