PROJECT: The Olfactory System: Studies in Gene Regulation and Genome Evolution
INVESTIGATOR: Robert Lane (MB&B)
The olfactory system is a primal sensory system for most animals. The sense of smell is required in order for animals to detect food sources, avoid predators, and find mates. To accomplish this, the olfactory system must distinguish among thousands of odorants in the environment. Moreover, the olfactory system must be specialized for the niche and lifestyle of each species. Our laboratory is concerned with these two fundamental issues: How does the olfactory system distinguish odorants, and how does the olfactory system evolve individual species capabilities?
The ability to detect and distinguish among thousands of environmental odorants is based on a combinatorial recognition system. Encoded in the animal's genome are about a thousand odorant receptor (OR) proteins that are expressed in olfactory sensory neurons. A specific "smell" is coded in the brain by a specific combination of receptor proteins that get stimulated by the unique combination of odorant chemicals elicited by that smell. The smell of "lemons", for example, would result from a specific combination of OR proteins that become stimulated upon binding the specific set of inhaled chemicals emitted from a lemon. But, how does the brain know which specific set of OR proteins are active? The key to this ability is an critical organizing principle in the olfactory system: each sensory neuron transcribes only one OR protein, thus, each olfactory sensory neuron is dedicated to report the response of a single OR. The problem of how smells are recognized and interpreted by the animal can be reduced to the question of how sensory neurons develop their OR-specific phenotypes. A major part of the research in our laboratory is focused on this remarkable gene regulatory problem: what are the molecular mechanisms underlying a sensory neuron's ability to exclusively express a single OR protein. We are using molecular biology, genetics, comparative genomics, and bioinformatic techniques to investigate gene regulation in the olfactory system.
If different animals, even closely-related ones, possess an olfactory system adapted to their own unique niche and lifestyle, then there are two (not mutually exclusive) possibilities: either the repertoires of odorant receptor genes are species-specific (i.e., there are differences in peripheral responsiveness), or the post-synaptic wiring patterns are species-specific (i.e., there are differences in how the same combinations of odorants are responded to centrally). We have focused our attention on the former possibility, and in particular, on the evolution of candidate pheromone receptor gene repertoires in various species. Pheromones are chemicals emitted by members of one's own species that elicit stereotyped reproductive/social responses. Most mammals have a "second nose", the vomeronasal organ (VNO), whose functions have been tightly associated with mating and social behaviors. One family of odorant receptor proteins (therefore, candidate pheromone receptor proteins) expressed in the VNO is the V1R gene family. Since mating and social behaviors are often exquisitely species-specific, we speculate that the putative pheromone receptors of the VNO (e.g., V1Rs) will represent an extreme in species-specific adaptation. With this in mind, a second major aspect of our research is applying tools of molecular biology, comparative genomics, and bioinformatics to investigate how V1R repertoires become different during speciation.
PROJECT: Transcriptional regulation of rRNA metabolism related genes in yeast
INVESTIGATOR: Michael McAlear (MB&B)
The coordinated expression of specific gene sets is essential for the execution of many cellular processes ranging from cell division to cellular differentiation. The recent development of microarray technologies has enabled researchers to begin to characterize and define these networks. By analyzing a wide range of transcriptional datasets from the budding yeast S. cerevisiae, the McAlear laboratory has discovered one of the largest co-regulated gene sets ever identified in yeast. They have found that over 200 yeast genes (nearly 4% of the entire genome) are coordinately expressed, and that the gene products function in the ribosomal and rRNA biosynthesis (RRB) pathway. The members of this RRB regulon are distinct from the ribosomal protein (RP) regulon, in that they do not encode for structural components of the ribosome itself, rather they encode factors required for rRNA biosynthesis ranging from transcription factors to RNA helicases. Microarray experiments performed with Wesleyan's Affymetrix Genechip facility have confirmed and extended this discovery, revealing that the RRB genes are co-regulated across a wide range of conditions.
Computational analysis of the RRB gene promoters has revealed that they are highly enriched for two short (11 to 13 basepair) consensus motifs know as PAC and RRPE. Deletion analysis has revealed that these motifs are important for the transcriptional regulation of RRB regulon members. Current experiments are aimed at identifying the trans factors that interact with these motifs, thereby regulating the coordinated expression of a large set of metabolically active genes.
PROJECT: Proteomic and Cell Biological Analysis of Fission Yeast Telomere-Associated Factors
INVESTIGATOR: Mark Flory (MB&B)
Our group is interested in identifying and characterizing telomere-associated factors in the model system of fission yeast (Schizosaccharomyces pombe) and understanding their function in the global context of cell cycle-dependent chromosome dynamics. We are specifically focusing on Ccq1p (coiled-coil protein quantitatively enriched) that we previously demonstrated to associate with the telomere during interphase, and segregating chromatin during mitotic anaphase. Our analysis has also shown that, together with the centrosome component Pcp1p, Ccq1p facilitates the critical but mysterious event of meiotic telomere-centrosome clustering. Our work provided one of the first physical mechanisms for this important meiotic event, known generally as chromosomal "bouquet" formation, which is conserved from yeast to humans and appears based on others work to be required for meiotic recombination. Our work also indicates that, in addition to its meiotic role, Ccq1p normally provides a critical protective role to the cell by preventing telomeric degradation and anaphase bridging during vegetative growth. In our current work we are using a series of chromosomally-integrated, epitope-tagged alleles of ccq1 to enrich for relevant binding partners via affinity chromatography. Preliminary proteomic analysis via mass spectrometry of affinity-enriched complexes suggests a role for Ccq1p in cell cycle control. We are complementing our proteomic analysis by combining ccq1 knockout alleles with other loss-of-function mutants to define genetic interactions. Live-cell epifluorescence microscopy of integrated fusions of Ccq1p and identified proteins to fluorophores such as GFP and RFP is also proving extremely useful in characterizing function. Finally, we are also engaged in a number of both internal and external proteomic collaborations including analysis of translational mechanisms in the budding yeast Saccharomyces cerevisiae (Weir and Rice Laboratories, Wesleyan University), detection of label-accessible human hemoglobin residues (Mukerji Laboratory, Wesleyan University) and dissection of various protein complex interactions in S. pombe and Z. mays (Cande Laboratory, UC Berkeley).
PROJECT: Transcriptional silencing in yeast
INVESTIGATOR: Scott Holmes (MB&B)
The research group of Dr. Scott Holmes focuses on the mechanism of transcriptional silencing in yeast. Silencing controls the developmental program of yeast, and involves a modification of local chromatin structure to repress gene expression. Once established this repressive chromatin structure is inherited in an epigenetic pattern. Several factors have been identified that are required for silencing in yeast; some of these are specifically dedicated to controlling transcription, while others have more general activities in the cell. Dr. Holmes is currently using a variety of methods to determine the mechanism of silencing.
The Holmes Lab has examined the establishment of silencing at yeast telomeres using inducible expression of the Sir3 silencing factor. Silencing is assayed by a sensitive real-time PCR assay. Using this system it was found that silencing is established only when cells are allowed to progress through mitosis. This requirement can be abolished by inactivation of proteins proposed to establish boundaries between active euchromatin and inactive heterochromatin. Current experiments combine chromatin immunoprecipitations with real-time PCR to determine how these factors influence the assembly of silencing complexes and histone modifications.
An independent project focuses on a novel form of silencing mediated by the REP3 element, a cis-acting DNA sequence required for proper segregation of the 2-micron circle, a naturally occurring plasmid in yeast. REP3 silencing requires two other plasmid-encoded proteins, Rep1 and Rep2. Using a genetic assay it was recently shown that the Rep1 protein interacts with the Sir silencing factors and with Scc1, a protein mediating chromosomal cohesion. The Holmes group is using fluorescence microscopy to examine the relative localization of these factors within the yeast cell, and have initiated a proteomics study to comprehensively determine Rep1 and Rep2 interacting proteins.
PROJECT: The roles of interneurons in the organization of cortical activity
INVESTIGATOR: Gloster Aaron (Biology)
Inhibitory interneurons in the mammalian cortex are at least necessary for regulating the spatial and temporal propagation of action potential activity that constitutes the substrate of conscious sensation, discrimination, and decision making.
The differences between different classes of inhibitory interneurons in the cortex are so great that their respective roles in sculpting cortical activity must also be different. In order to differentiate and understand these different roles there are certain measurements that are necessary, such as: (1) the network activity of the cortex itself; (2) identification of particular classes of interneurons within that measured network. Measurement (1) is so far impossible in the ideal sense--one can't measure the activity of every single neuron in the cortex. Using optical measurements from a slice of cortex, however, we can measure the activity of hundreds of single neurons that may comprise functional microcircuits within the cortex. These slices of cortex are taken from transgenic GFP mice that have been engineered so that only particular classes of interneurons express GFP, thus optically distinguishing them from the rest of the network. In this arrangement we can produce minutes-long optical "movies" of cortical slices where the spontaneous activity from hundreds of neurons are recorded along with the identities of those neurons belonging to a particular interneuronal class. We can also control the activity of select GFP-labeled interneurons with intracellular whole-cell patch-clamp recordings and see how the activity of those interneurons may affect the network surrounding them. The gigabyte-sized movies acquired are analyzed by several processors so that a rapid identification of which neurons are active and participating in non-random network dynamics are identified. We can then aim our intracellular electrodes at those neurons identified by the computer and further dissect functional relationships between interneurons and other neurons in the network that comprise these dynamic cortical circuits.
PROJECT: Studies of mouse embryogenesis using embryonic stem cells
INVESTIGATOR: Laura Grabel (Biology)
Dr. Laura Grabel and her research group use mouse embryonic stem (ES) cells to investigate the role of signaling molecules in early mammalian development. These cells can differentiate in vitro into a variety of cell and tissue types, providing a convenient cell culture model for the mouse embryo. Current studies in the Grabel group focus on the role of signals encoded by the Hedgehog gene family. They and their colleagues (in Dr. Andrew McMahon's group at Harvard University) have generated mutant ES cells lines deficient for genes encoding members of the Hedgehog signaling pathway, including Indian Hedgehog. Analysis of the differentiation of these mutant cell lines suggests a role for this pathway in neurectoderm differentiation and yolk sac vasculogenesis. For each of these differentiation pathways, a host of molecular markers have been identified, and their expression pattern provides a readout of the extent of differentiation of the mutant cell lines.
The technology available today, including RT-PCR and in situ hybridization analysis, permits members of the Grabel group to study, one at a time, the expression of only a handful of these markers. More complete characterization of the mutant cell lines would be greatly facilitated by the more comprehensive analysis of gene expression patterns that can be observed using microarray analysis. In a single experiment they could investigate the expression of hundreds of genes associated with a given cell or tissue type. Comparison of total gene expression profiles for mutant versus wild type cell lines could also reveal additional roles for Hedgehog signaling not identified in the Grabel group's initial analysis. The feasibility of this approach is underscored by the observation that the mouse ES system has already been successfully employed for microarray analysis, since large amounts of synchronously differentiating material can be obtained (see Kelly and Rizzino, Mol Reprod Dev 56:113-23, June 2000).
PROJECT: Origins of bacterial species
INVESTIGATOR: Fred Cohan (Biology)
Dr. Fred Cohan and his research group are investigating the origins of bacterial species, using a laboratory microcosm as a model system. When a single clone of Bacillus subtilis is cultured for several weeks, it inevitably evolves into a diversity of ecologically distinct populations. Dr. Cohan and his students are studying the environmental conditions that facilitate the origins of such populations. Dr. Cohan is also studying the degree to which an adaptive radiation of one clone into multiple populations is a repeatable process, so he is proposing to characterize the genetic changes underlying the ecological divergence among populations. Dr. Cohan proposes to identify genome-wide differences in gene expression among populations using gene chip technology.
Dr. Cohan and his research group have developed a sequence-based, "community phylogeny" algorithm for estimating the number of ecotypes (ecologically distinct populations with species-like characteristics) in a bacterial community, and identifying them. This is a maximum likelihood approach that finds the number of ecotypes, as well as the rate of periodic selection (ecotype-wide purging of diversity, which occurs in taxa with rare recombination) and rate of speciation, which yield the pattern of sequence diversity in a natural community. This approach yields a way to estimate the ecological diversity within a community without having to make any assumptions about the level of sequence diversity typically found within an ecologically distinct population. Dr. Cohan is currently estimating the ecotype diversity within five bacterial communities and taxa.
PROJECT: Molecular dynamics and Monte Carlo computer simulation; Structure, dynamics and solvation of nucleic acids and proteins INVESTIGATOR: David Beveridge (Chemistry)
PROJECT: Molecular genetic analysis of protein export pathways in bacteria
INVESTIGATOR: Don Oliver (MB&B)
PROJECT: Developmental and comparative studies of Hox gene function
INVESTIGATOR: Ann Burke (Biology)
PROJECT: Information Theoretic Analysis of Drosophila cDNAs
INVESTIGATORS: Michael Weir (Biology) and Michael Rice (Computer Science)
An important ongoing effort of the Berkeley Drosophila Genome Project is to assemble the sequences for a large set of Drosophila cDNAs. By comparing these sequences with their corresponding genomic sequences, we have computed a set of over 24,000 splice sites.
We are using information-theoretic approaches to analyze these splice sites, with relational databases as an framework for our analysis. We have found that the sequence conservation near splice sites depends upon the lengths of introns and exons in their neighborhoods. By examining sets of splice sites where the spliceosome machine is considered to be strained, we are gaining insights into the mechanisms that govern splicing.
We are also using information-theoretic approaches to study how the translation of mRNAs is initiated in eukaryotes. This work is in a preliminary stage.