No description available.

The olfactory sensitivity of five male CD-1 mice (Mus musculus) for six amino acids was determined using an operant conditioning paradigm. All animals significantly distinguished dilutions as low as 0.01 mM L-cysteine, 3.3 mM L-methionine, 10 mM L-proline, 0.03 mM D-cysteine, 0.3 mM D-methionine and 10 mM D-proline from the odorless solvent, with individual animals displaying even lower detection thresholds. Among the three different L-forms of the amino acids the mice were most sensitive for cysteine and least sensitive for proline, and among the three D-forms the animals displayed a lower sensitivity for D-proline compared to D-cysteine and D-methionine. A comparison between the present data and results obtained with other species showed that the CD-1 mice displayed a higher sensitivity than human subjects and spider monkeys with three (L-Cysteine, D-cysteine and L-proline) of the six amino acids. Results from this report support the idea that the number of functional olfactory receptor genes is not suitable to predict a species’ olfactory sensitivity.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 93-102). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [115]-133).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-157).

Thesis (Ph. D.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 92-102). Also available on the World Wide Web.

The development of an inexpensive, portable, stable, sensitive and selective biosensor for detection of odorants is a daunting task. Here, we hypothesized the development of a detector layer composed of the protein groups; the olfactory receptors (ORs) and the odorant binding proteins (OBPs), known to bind odorants in animal sensing. We report the design of 13 OR gene-vector constructs, and their subsequent transformation into Escherichia (E.) coli (BL21 (DE3)-STAR-pLysS) strain. Moreover, we report the expression of several ORs into an in vitro wheat germ extract using three separate detergent mixes for protein solubilization.   In addition, we describe the design of an odorant binding protein from the Aenopheles gambiae mosquito PEST strain (OBP-PEST) gene-vector construct under an IPTG (Isopropyl β-D-1-thiogalactopyranoside) inducible promoter. OBP-PEST was heterologously expressed in E.coli with an 8 amino acid long sequence (WSPQFEK) attached C-terminally, via a thrombin cleavage site and a flexible linker (GSSG). The WSPQFEK sequence, commonly referred to as a Strep-tag, enabled subsequent affinity chromatography purification of the protein, via binding to an engineered Streptavidin equivalent. Surprisingly, the OBP-PEST was found to contain a signal sequence leading to its truncation and secretion when expressed in E.coli.   Biophysical analyses were established using Circular Dichroism (CD) for the analysis of two lipocalins: Beta-lactoglobulin (BLG) and OBP-PEST. We studied the solubility, refoldability and the conformational transitions of BLG, as a result of change in solvent, pH and temperature. The secondary structure of OBP-PEST and its thermal stability was investigated.   In conclusion, this thesis work has enabled biophysical analyses of OBP-PEST and future analogs of interest to the development of a stable protein detector layer. Although further experiments are needed to fully characterize the biophysical properties, and to find odorant substrates of OBP-PEST, it was found to be a suitable alternative to ORs in a biosensor detector layer application. More importantly, an inherent OBP-PEST signal sequence was found to mediate protein secretion when expressed heterologously in E.coli. To the best of our knowledge this is the first lipocalin discovered to be secreted upon heterologous expression in E.coli.   We hypothesize that this signal peptide could be used as a means for targeted secretion and, hence, efficient protein purification.

Mammalian olfaction requires the enduring expression of a single olfactory receptor (OR) gene for the life of each sensory neuron. This is due to the fact that OR proteins play multiple roles in the coherent perception of odors, first by sensing molecular cues from the external environment, and by directing the wiring of neuronal projections faithfully from the peripheral sensory neurons to the brain. Both of these processes require singular and stable OR expression in olfactory sensory neurons (OSNs. The transcription factor Atf5 has previously been shown to enforce these modes of expression, through a process that requires the unfolded protein response (UPR). The work presented in this thesis deciphers how Atf5 enables proper OR expression and neuronal function in the olfactory system. We identify the developmental window in which UPR is activated, and provide evidence that Atf5 protein expression coincides with the assembly of a multi-chromosomal enhancer hub that drives singular and robust OR transcription, opposing a model in which precocious polygenic OR transcription initiates UPR. Further, we show that Atf5 directly regulates a collection of genes that facilitate proper OR trafficking, axonogenesis, as well as transcription factors and chromatin modifiers, which we propose to be involved in stable OR expression and neuronal maturation. Finally, we find that Atf5 has a special role in the olfactory system that cannot be replaced by its ubiquitously expressed homologue, Atf4, and that this is due to a requisite interaction between Atf5 and the bZIP transcription factor Cebpγ, and potentially other transcription factors known to be critical for olfactory function.

No description available.

<p>Sensory neuron diversity is a common theme in the animal kingdom. It provides the cellular infrastructure that supports the accurate perception of the external world. Among all sensory systems, the olfactory system demonstrates an extreme in the extraordinarily diversified neuronal classes it holds. The system-wide cellular diversity is in sharp contrast with the individual specialization of olfactory receptor neurons (ORNs) per se. How the nervous system, particularly the olfactory system, uses limited genetic information to generate a huge variety of neurons with distinct properties remains elusive. </p><p>The adult Drosophila olfactory system is an excellent model to address this question due to its conserved organizational principles and reduced complexity. The fly olfactory appendages contain 50 ORN classes, each of which expresses a single receptor gene from a family of ~80 genes. Stereotyped clusters of 1-4 ORN classes define about 20 sensilla subtypes, belonging to 3 major morphological types. All cellular components within a sensillum are born by a single sensory organ precursor (SOP) via asymmetric divisions. The molecular mechanisms that determine SOP differentiation potentials to develop into distinct sensilla subtypes and the associated ORN classes are unknown.</p><p>From a genetic screen, we identified two mutant alleles in the rotund (rn) gene locus, which has a critical function in diversifying ORN classes. Rn is required in a subset of SOPs to confer novel sensilla subtype differentiation potentials from otherwise default ones within each sensilla type lineage. In rn mutants, ORNs in rn-positive sensilla subtypes are converted to lineage-specific default rn-negative fates, resulting in only half of the normal ORN diversity. This work is described in Chapter 2.</p><p>Based on an unbiased time-course transcriptome analysis, we discovered two critical downstream targets of Rn, Bric-à-brac (Bab) and Bar. In light of the knowledge about leg development, we found these genes, along with Apterous (Ap) and Dachshund (Dac), are part of the conserved proximal-distal (PD) gene network that play a crucial role in patterning the antennal precursor field prior to proneural gene-mediated SOP selection. Interactions between these PD genes under the influence of morphogen gradients separate the developing antennal disc into 7 concentric domains. Each ring is represented by a unique combination of the aforementioned transcription factors, coding the differentiation potentials for a limited number of sensilla subtypes. Genetic perturbations of the network lead to predictable changes in the ratios of different sensilla subtypes and corresponding ORN classes. In addition, using CRISPR/Cas9 technology, we were able to add tags to specific rn isoforms in the endogenous locus, and show positive regulation of Bab and negative regulation of Bar by the direct binding of Rn to the promoters in vivo. This work is presented in Chapter 3.</p><p>We proposed a three-step mechanism to explain ORN diversification, starting from pre-patterning of the precursor field by PD genes, followed by SOP selection by proneural genes, and ended with Notch-mediated neurogenesis. The final outcomes are greatly determined by the pre-patterning phase, which may be modified during evolution to compensate special olfactory needs by individual species. In our model, each step serves a single purpose, which displays context-dependent functions. By changing contexts, reassembly of the same logical steps may guide neuronal diversification in parallel systems with completely different identities. This step-wise mechanism seems to be a common strategy that is used by many other systems to generate neuronal diversity.</p> / Dissertation