Richard Axel

A novel multigene family may encode odorant receptors: A molecular basis for odor recognition

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.

The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain

The isolation of clones encoding the human surface protein T4, and the expression of the T4 gene in new cellular environments, have enabled us to examine the role of this protein in the pathogenesis of AIDS. Our studies support a mechanism of AIDS virus infection that initially involves the specific interaction of the AIDS virus with T4 molecules on the cell surface. This association can be demonstrated on T4+ transformed T and B lymphocytes as well as epithelial cells. Furthermore, the presence of T4 on the surface of all human cells examined is sufficient to render these cells susceptible to AIDS virus infection. Our data suggest that the T4-AIDS virus complex is then internalized by receptor-mediated endocytosis. Finally, we find that the T4 gene is expressed in the brain as well as in lymphoid cells, providing an explanation for the dual neurotropic and lymphotropic character of the AIDS virus. In this manner, a T lymphocyte surface protein important in mediating effector cell-target cell interactions has been exploited by a human retrovirus to specifically target the AIDS virus to populations of T4+ cells.

Visualizing an Olfactory Sensory Map

We have developed a genetic approach to visualize axons from olfactory sensory neurons expressing a given odorant receptor, as they project to the olfactory bulb. Neurons expressing a specific receptor project to only two topographically fixed loci among the 1800 glomeruli in the mouse olfactory bulb. Our data provide direct support for a model in which a topographic map of receptor activation encodes odor quality in the olfactory bulb. Receptor swap experiments suggest that the olfactory receptor plays an instructive role in the guidance process but cannot be the sole determinant in the establishment of this map. This genetic approach may be more broadly applied to visualize the development and plasticity of projections in the mammalian nervous system.

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk−, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk+ clones with a frequency of 10−6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk+ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk− phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10−3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.

Topographic organization of sensory projections to the olfactory bulb

The detection of odorant receptor mRNAs within the axon terminals of sensory neurons has permitted us to ask whether neurons expressing a given receptor project their axons to common glomeruli within the olfactory bulb. In situ hybridization with five different receptor probes demonstrates that axons from neurons expressing a given receptor converge on one, or at most, a few glomeruli within the olfactory bulb. Moreover, the position of specific glomeruli is bilaterally symmetric and is constant in different individuals within a species. These data support a model in which exposure to a given odorant may result in the stimulation of a spatially restricted set of glomeruli, such that the individual odorants would be associated with specific topographic patterns of activity within the olfactory bulb.

Transformation of mammalian cells with genes from procaryotes and eucaryotes

Abstract We have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist. The addition of a purified viral thymidine kinase (tk) gene to mouse cells lacking this enzyme results in the appearance of stable transformants which can be selected by their ability to grow in HAT. These biochemical transformants may represent a subpopulation of competent cells which are likely to integrate other unlinked genes at frequencies higher than the general population. Co-transformation experiments were therefore performed with the viral tk gene and bacteriophage ΦX174, plasmid pBR322 or the cloned chromosomal rabbit β-globin gene sequences. Tk + transformants were cloned and analyzed for co-transfer of additional DNA sequences by blot hybridization. In this manner, we have identified mouse cell lines which contain multiple copies of 4)X, pBR322 and the rabbit β-globin gene sequences. The ΦX co-transformants were studied in greatest detail. The frequency of co-transformation is high: 15 of 16 tk + transformants contain the ΦX sequences. Selective pressure was required to identify co-transformants. From one to more than fifty ΦX sequences are integrated into high molecular weight nuclear DNA isolated from independent clones. Analysis of subclones demonstrates that the ΦX genotype is stable through many generations in culture. This co-transformation system should allow the introduction and stable integration of virtually any defined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.

A Spatial Map of Olfactory Receptor Expression in the Drosophila Antenna

Insects provide an attractive system for the study of olfactory sensory perception. We have identified a novel family of seven transmembrane domain proteins, encoded by 100 to 200 genes, that is likely to represent the family of Drosophila odorant receptors. Members of this gene family are expressed in topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. Sensory neurons express different complements of receptor genes, such that individual neurons are functionally distinct. The isolation of candidate odorant receptor genes along with a genetic analysis of olfactory-driven behavior in insects may ultimately afford a system to understand the mechanistic link between odor recognition and behavior.

A novel family of genes encoding putative pheromone receptors in mammals

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct sensory organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of about 30 putative receptor genes. Sequence analysis indicates that these genes comprise a novel family of seven transmembrane domain proteins unrelated to the receptors expressed in the MOE. Moreover, the expression of each member of the gene family is restricted to a small subpopulation of VNO neurons. These genes are likely to encode mammalian pheromone receptors.

Allelic inactivation regulates olfactory receptor gene expression

We suggest a model in which a hierarchy of controls is exerted on the family of odorant receptor genes to assure that a sensory neuron expresses a single receptor from a family of 1000 genes. We propose that a cis-regulatory element directs the stochastic expression of only one gene from a large array of linked receptor genes. Moreover, only one allelic array encoding multiple receptor genes is active in an individual neuron. We demonstrate that in a neuron expressing a given receptor, expression derives exclusively from one allele. In addition, we observe that alleles encoding the odorant receptors are replicated asynchronously, a phenomenon consistently associated with allelic inactivation. This model, involving inactivation of one allelic array and cis control of the active array, provides a mechanism such that individual neurons express one or a small number of receptors.