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The olfactory receptors (ORs), which are G-protein coupled receptors, number more than 1,000 and comprise the largest gene family in the mammalian genome. ORs are expressed both monogenically and monoallelically in olfactory sensory neurons (OSNs) and the mechanism that controls their regulation is largely unknown. ORs reside in constitutive heterochromatin and selection of one OR and from one allele occurs stochastically. To ensure singular monoallelic OR expression, a negative feedback mechanism is elicited by the first selected OR to suppress non-selected ORs in a given neuron. Here we describe results for mice with a 'monoclonal' nose that express one OR, M71,in 95% of all mature OSNs. The M71 transgene suppresses expression of endogenous ORs, and remaining endogenous expression is mostly restricted to immature neurons in the olfactory epithelium. We show that the endogenous OR repertoire are expressed prior to suppression by M71 transgene expression, contrary to current models. When we introduced a second transgene into M71 mice that expressed another OR in most mature OSNs, OSNs uncharacteristically expressed both of the ORs. We hypothesize that unresolved OR competition compromised the neuron's ability to express only one receptor. We further show that suppression of endogenous ORs by M71 is not reversible, and that M71 does not need to be continuously expressed for endogenous ORs to remain suppressed. In these experiments, we have engineered OSNs to express more than one OR in an OSN at a time, which is normally a low probability event. We believe that these experiments reveal the existence of a backup pathway that ensures only one OR will be expressed per neuron. It is possible that mammals have evolved similar mechanisms in other examples of stochastic, monoallelic gene expression, such as in random X-chromosome inactivation or lymphocyte receptor expression.

Background and Objective: The Arabian oryx (Oryx leucoryx) was hunted to near extinction in from the 1950s to the 1970s. Thus, the oryx ranks among the rarest mammals in the world. Qatar currently has ~800 oryx and has donated numerous oryx to breeding programs around the world. As such, Qatar may be a significant source of genetic diversity to the worldwide oryx population. The primary purpose of this project was to provide a genome sequence and Single Nucleotide Polymorphism (SNP) data in hopes it can be used for improving breading strategies by maintaining as much genetic diversity as possible. Methods: A DNA sample was provided from an oryx male in the Wabra Wildlife Preservation in Qatar. The animal's whole genome was sequenced using next-generation sequencing approach. After assembling the contigs, we utilized a 67bp kmer and ~2.4B paired 100bp (~80X coverage) reads from the Illumina HiSeq. These reads were distributed across libraries ranging in size from 300-1200bp for paired-end and 2000-5000bp for mate-pair libraries. Genome physical coverage by mate-pair libraries was approximately 15X. De novo gene prediction was conducted on scaffolds >500bp. SNPs were also detected. Results: Predicted genome size of ~3Gb (similar to other Mammals), a scaffold N50 of ~300kb, an assembly spanning ~2.5Gb of the genome which is likely >90% of euchromatic sequence. The sequence is distributed across 36,964 scaffolds greater than 500bp. We detected ~1M SNPs between parental alleles, which is significantly fewer than in other "rare" animals such as the giant panda. Conclusion: The initial analysis of polymorphisms suggests a relatively high level of inbreeding, and further study will be needed to clarify whether this is only in certain herds or a worldwide issue.

Each olfactory sensory neuron in the mammalian nose selects just a single member of the large odorant receptor (OR) gene family. The core processes regulating OR selection may involve mechanisms that limit initial OR transcription and once a single OR is chosen, repress subsequent activations. We have used a genetic strategy to monitor the transcriptional permissiveness of the OR gene P2 by inserting an exogenous promoter, the tetracycline-dependent transactivator responsive promoter (teto), into its start site through homologous recombination. We observe that the OR locus limits the expression of the teto: repressing it outside of the wild type P2 zone while allowing sporadic activation from within its zone. Staged conditional expression experiments reveal that the receptor locus becomes fully repressed over time and that this repression does not require the receptor's open reading frame. Further, the exogenous promoter is inhibited by an OR transgene that similarly suppresses the endogenous rec...

Many animals have evolved mechanisms to withstand the harsh desert environment, characterized by extreme high temperatures and scarce water supplies. The Arabian Camel, and the Arabian Oryx are valued economically and culturally. These animals can survive for several days without food or water. As the Arabian peninsula undergoes rapid and vast industrial changes, it is increasingly important to understand the biological aspects of these animals. From the unicellular microbes to the sophisticated multi-cellular animals, sensing the chemical composition of the surrounding environment is essential for survival. The vertebrate chemosensory receptors genes, which are members of the seven transmembrane -helical G-protein coupled receptors (GPCRs), are encoded by six different multigene families. Four of these genes encode receptor proteins for sensing odors. The olfactory (odorant) receptors (ORs) are predominately expressed in the sensory neurons of the main olfactory epithelium, and can sense either water-soluble (class I) or volatile (class II) molecules. Furthermore, certain OR genes are expressed in non-olfactory tissues, such as brain, kidney, testis, and placenta. Being adapted to very harsh conditions with elevated temperature, scarce water supply and limited vegetation, we hypothesize that desert animals have evolved the ability to detect water either from volatiles liberated by water in the environment or through the blooming of short-lived vegetation, via their olfactory systems. To explore this possibility we have undertaken the study of the Camel OR genes. We identified approximately one hundred candidate OR genes, all of which are ortholgos to OR genes in other mammals and most closely related to those of the Equus caballus. Preliminary analysis revealed an enrichment in OR gene family 2/13, found in the highest proportion in aquatic animals, as compared to other mammals. This finding provides the intriguing suggestion that desert animals have evolved specific OR genes to adapt to the desert ecosystem. We are currently working to identify the complete OR gene repertoire in the Camel, and to identify and characterize the OR subgenome in other desert animals such as the Arabian Oryx.

Smell is an essential sense that allows animals to find food and mates while avoiding predators. In humans smell is considered an aesthetic sensory modality, but olfactory disorders may presage neurological disease including Parkinson's, Alzheimer's, and schizophrenia. The odorant receptors (ORs) comprise the largest gene family in mammals and endow an animal with the ability to smell. Critical to the development and function of olfaction is the regulation of OR gene transcription, with each sensory neuron selecting just one OR for expression, at random, from only one allele. While recent experiments have brought the outlines of this remarkable process into focus,the core mechanism has remained obscure. Using a genetic approach in mice we present data that supports a model of single OR gene choice initiated by infrequent, stochastic transcriptional activation and governed by feedback suppression mechanism.In the face extreme requirements for diversity, cells and organisms have evolved stochastic p...