J Gregor Sutcliffe, PhD - US grants

This is a proposal to continue ongoing studies to describe the structures of rat brain mRNAs, identify and characterize their protein products and characterize cis acting elements which may regulate their expression. 1) We will continue our characterization of the 1B236/MAG gene products. These studies will include refining structural information about the relationship between 1B236 and myelin- associated glycoprotein (MAG), investigation of the cell types in which particular alternatively spliced RNA products are formed, assignment of the membrane orientation of the 1B236/MAG protein, and search for an endogenous ligand for this protein which shares sequence identities and domain structures with immunoglobulin-type receptor molecules. 2) We will further study the distribution and transcription during development by Pol II and Pol III of ID elements, and isolate and characterize the mouse and monkey ID analogues. 3) We will study the enhancer properties of ID elements in neuronal and kidney cell cultures. 4) We will study the relationship between ID enhancer phenotypes and Pol III template activity by saturation point mutagenesis. 5) We will isolate full-length cDNA clones of rat mRNAs exclusive to or highly enriched in the brain compared to liver and kidney that exhibit regional differences in their concentration within brain, cross hybridize to analogous mouse and monkey brain mRNAs, and have postnatal developmental onset of expression. 6) We will determine the nucleotide sequences, map the 5' ends and investigate alternative forms of these mRNAs and then use the amino acid sequences deduced from their open reading frames to conduct computer searches and synthesize homologous peptides and fusion proteins. Antisera to these peptides will be used to describe the biochemical features and anatomical locations of the protein. 7) We will measure the relative portion of brain mRNA whose expression has postnatal onset.

We have developed the technique of subtractive hybridization as a powerful tool for isolating clones of mRNAs that are restricted to anatomically-defined subsets of neurons. Using this technique we have isolated the product of the rds neuronal degeneration gene. We propose studies to test a model for its function as an intralumenal adhesion molecule, to improve our understanding of the disposition of the rds protein in the outer segment disc membrane, to prove that inactivation of the rds protein is solely responsible for the rds phenotype, and to understand fully the nature of the rds mutagenic event and mutant protein expression. We have also isolated clones of mRNAs encoding a telencephalon-enriched, calmodulin-binding substrate for protein kinase C and a telencephalon-enriched Ca++, calmodulin-dependent protein kinase. To interpret the physiological significance of each, we will map their genes so as to learn whether they are the products of any known gene for which mutants have been characterized and we will also produce deliberate mouse mutants affected in their expression. To this end, we will develop ribozyme and homologous recombination gene knock-out methodologies. We will produce recombinant proteins for biochemical assessments of function, including proof that the kinase truly has that activity and a search for its natural substrates. We will investigate amino acid sequences that direct proteins to dendrites.

This is a proposal to continue ongoing studies to describe the structures of rat brain mRNAs, identify and characterize their protein products and characterize cis acting elements which may regulate their expression. 1) We will continue our characterization of the 1B236/MAG gene products. These studies will include refining structural information about the relationship between 1B236 and myelin- associated glycoprotein (MAG), investigation of the cell types in which particular alternatively spliced RNA products are formed, assignment of the membrane orientation of the 1B236/MAG protein, and search for an endogenous ligand for this protein which shares sequence identities and domain structures with immunoglobulin-type receptor molecules. 2) We will further study the distribution and transcription during development by Pol II and Pol III of ID elements, and isolate and characterize the mouse and monkey ID analogues. 3) We will study the enhancer properties of ID elements in neuronal and kidney cell cultures. 4) We will study the relationship between ID enhancer phenotypes and Pol III template activity by saturation point mutagenesis. 5) We will isolate full-length cDNA clones of rat mRNAs exclusive to or highly enriched in the brain compared to liver and kidney that exhibit regional differences in their concentration within brain, cross hybridize to analogous mouse and monkey brain mRNAs, and have postnatal developmental onset of expression. 6) We will determine the nucleotide sequences, map the 5' ends and investigate alternative forms of these mRNAs and then use the amino acid sequences deduced from their open reading frames to conduct computer searches and synthesize homologous peptides and fusion proteins. Antisera to these peptides will be used to describe the biochemical features and anatomical locations of the protein. 7) We will measure the relative portion of brain mRNA whose expression has postnatal onset.

One of the more remarkable scientific trends during the 1980's has been the mutual reciprocal movements of molecular biologists into questions of neuroscientific interests and neuroscientists, particularly those interested in human neurological disorders having a genetic basis and those interested in the structural parameters of neural function, into questions of gene and mRNA structure. Studies of the nervous system may represent perhaps the major intellectual and experimental endeavor of biological science over the next few decades. The consequences of this endeavor will have profound effects on the way human society perceives itself. The recent inaugural MOLECULAR NEUROGENETICS meeting combined presentations about vertebrates and invertebrates, cell lineages and cell interactions, cell biology, pharmacology, electrophysiology, biochemistry, and descriptions of the genetics underlying several neuropathologies. This application seeks support for the next two FASEB Summer Research conferences on MOLECULAR NEUROGENETICS.

The goal of this project is to produce an integrated map of murine chromosome 16. This map will correlate the positions of transcribed regions with the existing genetic map and produce a physical map of restriction endonuclease cleavage sites spanning the entire chromosome. The map will allow us to answer questions of gene number, overlap in the sets of expressed genes, gene clustering, correlation between physical and genetic maps, and chromosome architecture. The integrated map will be used to isolate the locus for the mouse cerebellar development gene weaver(wv) and the mouse region homologous to that responsible for Down's syndrome. 1. We will assemble a nearly complete mouse chromosome 16-specific genomic library that can be rapidly and repeatedly analyzed by hybridization. 2. We will identify clones of expressed sequences and define their patterns of expression. Of special interest will be sequences expressed in the CNS. 3. To place these sequences on the genetic map, clones of expressed regions will be used to identify RFLP's that distinguish strains of mice. These RFLP's will be used to establish linkage of the cloned expressed sequences with known markers. 4. These cloned sequences will be used to determine a large scale restriction map of chromosome 16. 5. The genetic and transcription maps will be used to identify candidate clones of the wv locus. These will be used to examine mutant ice to identify the wv gene. The wv gene products will be characterized. 6. The integrated maps will be studied with reference to the syntenic region of human chromosome 21 to identify candidates for the critical genes whose aneuploidy is responsible for Down's Syndrome.

We propose to build a database of quantitative information concerning the expression of the 10,000 to 20,000 most prevalent mouse brain mRNAs in different anatomical sites within the brain, at several developmental stages, in peripheral tissues, and in response to physiological and behavioral paradigms. From the data, to be collected by a newly developed PCR-based methodology conceptually similar to but more exhaustive than differential display, we will recognize a few thousand differentially expressed or physiologically regulated mRNAs. We will determine partial sequences of 900 differentially expressed or regulated mRNAs, which will provide an identity tag for each and lead to the identification of novel proteins. Specific PCR primers will be used to establish expression profiles at higher degrees of precision and for SSCP-PCR amplification of genomic DNA from a panel of 94 interspecific backcross mice so as to determine linkage of each PCR product, within a few cM, to markers already mapped on the panel. From these studies will emerge an annotated public database cataloguing PCR primer, PCR product length, relative amounts of the products in different tissues, partial nucleotide sequence and gene location in which genes identified by their differential expression patterns can be identified by scientists with particular developmental/physiological/ behavioral questions for in depth studies. Equally important, the genetic and expression pattern aspects will allow candidate genes for genetically determined disorders of the CNS and certain peripheral tissues to be recognized. The studies will answer questions of gene number and clustering of genes preferentially transcribed in specific anatomical and/or developmental windows, and provide a better understanding of patterns of gene expression, and thus will provide baseline data for a technology that is certain to become, because of its digital approach to total gene expression, a major analytic tool for discerning normal and defective physiological function.

oligonucleotides; nucleic acid sequence; peptide chemical synthesis; biomedical facility; nucleic acid chemical synthesis; protein sequence;

Microglia have been implicated as key players in the CNS inflammatory response to HIV infection. Inflammation leads ti astrogliosis and neuronal loss, pathologies that correlate with progressive AIDS dementia. Similar pathologies involving microglia are observed in several other degenerative brain conditions: trauma, abscess, focal ischemia, EAE, Wallerian degeneration, Down's syndrome and Alzheimer's disease. The information presently available about microglia in their quiescent and various activated forms is incomplete. We will 1. Utilize the newly developed molecular techniques of directional tag PCR subtractive hybridization and PCR-based differential display to identify cDNA clones of presently unknown mRNAs that are selectively expressed by populations of activated microglia. During the course of the proposed project, nearly all such molecules will be accounted. 2. Determine, using these newly identified mRNAs, the degree to which brain-resident microglia are related to other cells of the monocyte/macrophage lineage. 3. Elucidate the various ensembles of genes whose expression are activated in specific microglial subsets by different physiological and pathological processes such as viral infection, lentivirus glycoprotein exposure, and cytokine signalling. These data will suggest particular mRNAs whose protein products deserve functional characterization because of their potential roles in contributing to the pathophysiology that results in AIDS dementia.

The Computer Center was used for genetic sequence analyses pertinent to structure and function of neuronal cell nuclear components.

DESCRIPTION (provided by applicant): There are significant differences in the response phenotypes of various mouse strains in two models of behavioral despair, the forced swim and tail suspension tests. In both tests, mice cease to try to escape from an adverse situation, exhibiting increased immobility. Both tests have high value for predicting the therapeutic activities of antidepressant medications. We will study recombinant inbred (Rl) mice derived from parental mouse lines that differ in their immobility responses in despair tests and in their responses to antidepressants. The immobility data from the two tests in the presence or absence of medication will be correlated with the high-density haplotype data available online to identify chromosomal regions that harbor genes that contribute to the immobility phenotype: so-called Quantitative Trait Loci (QTLs). We will be especially interested in gender-specific QTLs because of the increased risk for depression among the female population. mRNA isolated from brain regions will be profiled to measure the relative concentrations of each mRNA. mRNAs that exhibit differences in concentrations between the two parental mice, are coinherited with each QTL, and which derive from the QTL chromosomal interval will be identified. Their products represent candidate despair-modifying proteins. Transgenic mice that either increase or decrease the expression of each candidate mRNA will be generated to verify that the quantitative behavioral trait is determined by the activity of the product of that candidate gene. The identities of the genes responsible for the QTs offer a powerful point of departure for the development of new pharmaceuticals to treat depression.