Lorraine Flaherty - US grants

The overall objective is to understand the genetics and genetic regulation of the Qa antigens encoded by the murine major histocompatibility complex. This complex has been a major concern to immunogeneticists for many years, initially because of its influence on tissue transplantation and more recently because of its involvement in determining the susceptibility to viral infections and successful cell-cell interactions during an immune response. The function of the Qa antigens and molecules has not been clearly defined although their unusual appearance on certain leukemia cells raises the possibility that they are involved in a normal or abnormal differentiation process. In this proposal, there are four specific aims: (1) to examine the allelism and polymorphism of genes within the Q subregion; (2) to determine the sites of recombination within and/or adjacent to the Q subregion, (3) to analyze the transcription and translation of Q subregions genes and (4) to study the control of the quantitative control of Q subregion gene products. Techniques to be used include molecular cloning and subcloning, isolation of genomic DNA and mRNA, Southern and Northern blot analyses, in vitro transcription and translation system, DNA mediated gene transfer, and DNA sequencing.

The long term goal of this proposal is to determine the cause of infantile polycystic kidney disease (PKD). In this laboratory, a new mouse model for autosomal recessive PKD has been found in the course of producing mutations with the drug, chlorambucil. This mutation has been preliminarily characterized as determining a severe infantile PKD; the gene has been called poly. Homozygous poly/poly mice die of extensive bilateral polycystic kidney disease at around 2 weeks of age. Most elements in the nephron appear to be affected. No other abnormalities have been noted. Heterozygous animals appear normal. This mutation is most likely due to a deletion of DNA since all other studied chlorambucil-induced mutations have been deletions. The following specific aims are proposed: (1) To characterize the PKD caused by the poly gene. Histological studies will be performed on both embryos and newborns. (2) To locate the chromosomal position of poly and determine if it is allelic to any known gene. Backcross and linkage crosses will be performed. DNA markers as well as morphological markers will be used for this purpose. More precise mapping will be performed once the gene is assigned to a particular chromosome. Allelism to nearby genes potentially affecting kidney function will be analyzed. (3) To walk into the gene by use of YAC libraries and positional mapping techniques. Approaches to this aim include the screening of YAC libraries, preparative pulsed-field gel electrophoresis, screening of cDNA kidney libraries, detection of CpG islands, zoo blots, Northern and Southern blotting, and direct positive selection of active kidney-expressed genes present on YACs. (4) To characterize the gene and determine its function. DNA sequencing and homology studies will be performed. Other animals mutants will also be screened for similar functional abnormalities. Finally, human families carrying a gene for infantile PKD will be studied by use of cross-hybridization techniques and Southern blot analysis.

The long term goal of this proposal is to develop a scheme for identifying and characterizing mammalian genes which cause developmental abnormalities. In the course of studying chlorambucil-induced mutagenesis in the mouse, we have found that chlorambucil (CHL) is a very efficient inducer of recessive visible mutations. Because CHL induces gross chromosomal abnormalities (deletions, translocations, etc.) instead of point mutations, the mutations caused by this drug can be easily mapped and characterized. We would like to use these mutations to positionally clone and characterize mammalian genes which cause developmental abnormalities. In this grant proposal, we would like to establish the parameters of the CHL mutagenesis system as well as produce a new series of recessive visible and developmental mutations in the mouse. Our specific aims are: (1) to determine the most efficient system for producing CHL mutations in mice, (2) to produce dominant and recessive visible mutations affecting development, (3) to map new CHL-induced mutations to specific chromosomal regions, and (4) to further characterize the usefulness of CHL as an inducer of specific mutations.

9322134 Mannella This award provides funds for renewal of a summer REU program held at the Wadsworth Center for Laboratories and Research (WCL&R) in Albany, NY. The research scientists who supervise the students in this program are also faculty in the Department of Biomedical Sciences of the State University of New York at Albany's School of Public Health. The WCL&R is a unique laboratory with state-of-the-art facilities, in which basic research programs have developed alongside mission-oriented public health laboratories. The research interests of the participating BMS faculty cover a broad range, with emphasis in molecular genetics, cell biology and structural biology. Eleven undergraduate students interested in research careers are recruited from colleges in the Northeast and from historically Black colleges in the South. Another 5 students participate in the program with stipend support from the WCL&R. Laboratory projects in basic biological research topics are chosen that are interesting, likely to yield results within the time-frame of the program, and allow for some degree of student development and independence. Weekly group meetings are used to discuss science- and career-related issues and to monitor student progress. The students make short oral presentations of their work in a symposium in the last week of the program, and also submit written reports before they leave, to stress the importance of communication skills within science. ***

The long term goal of this proposal is to develop a scheme for identifying and characterizing mammalian genes which cause developmental abnormalities. In the course of studying chlorambucil-induced mutagenesis in the mouse, we have found that chlorambucil (CHL) is a very efficient inducer of recessive visible mutations. Because CHL induces gross chromosomal abnormalities (deletions, translocations, etc.) instead of point mutations, the mutations caused by this drug can be easily mapped and characterized. We would like to use these mutations to positionally clone and characterize mammalian genes which cause developmental abnormalities. In this grant proposal, we would like to establish the parameters of the CHL mutagenesis system as well as produce a new series of recessive visible and developmental mutations in the mouse. Our specific aims are: (1) to determine the most efficient system for producing CHL mutations in mice, (2) to produce dominant and recessive visible mutations affecting development, (3) to map new CHL-induced mutations to specific chromosomal regions, and (4) to further characterize the usefulness of CHL as an inducer of specific mutations.

DESCRIPTION (Adapted from the Applicant's Abstract): A new autosomal recessive mutation jcpk (juvenile congenital polycystic disease) was generated by chlorambucil mutagenesis. In the homozygous condition, this mutation causes a severe polycystic disease (PKD) which is more severe than previously described mouse PKD mutants. Homozygotes are detectable as early as 4 days and have grossly enlarged kidneys. Histologically, homozygote kidneys are highly abnormal even at birth, with cysts appearing throughout the kidney. Extreme dilatations of the bile ducts and pancreatic ducts are often seen. A late onset glomerulocystic disease is present in approximately 25% of the heterozygotes. The investigators have mapped the jcpk mutation to mouse chromosome 10. jcpk has recently been found to be allelic the bck mutation. By an extensive backcross, a fine genetic map of the region around the jcpk locus has been made. This map should enable the investigators to positionally clone the jcpk locus. The Specific Aims of the proposal are to: (1) build a YAC and BAC contig of the region surrounding the jcpk locus; (2) identify candidate genes for this locus; (3) identify, clone, and characterize the jcpk gene; and (4) characterize the genetics of cystic disease in +/jcpk heterozygotes. Arrayed cDNA libraries will be used for some of these studies. In addition, genetic experiments involving DNA mismatch repair-deficient mice and transgenic mice will be conducted.

DNA microarrays provide a sensitive, comprehensive and highly efficient means for monitoring a cell's genetic program. Microarray technology is currently being exploited to determine global RNA expression levels under relevant genetic and environmental conditions. This technical advance, coupled with ever-increasing DNA sequence databases, allows for the rapid identification of functionally related genes, as well as new protein sequence motifs and functional domains. The profound impact that microarray analysis is having on the field of molecular genetics has been the subject of several recent review articles, including an entire supplement in Nature Genetics devoted to this technology. The instrument system proposed in this application, the Genetic Microsystems GMS 417 Arrayer and GMS 418 Array Scanner, will enable investigators at the Wadsworth Center to make use of this valuable technology under affordable conditions. This system was chosen for its quality, state-of-the-art design for producing consistently precise, high-density DNA arrays onto several surfaces, including glass microscope slides, and a confocal laser scanner with high resolution and sensitivity. The Genetic Microsystems Arrayer and Scanner will allow a diverse set of scientists to explore a wide range of biological questions. These include analyzing arrays of mammalian expressed sequence tags (ESTs) to investigate the genetic basis of kidney disease, memory and cerebellar development, identifying human genes important for degeneration and regeneration of olfactory mucosa, determining factors involved in DNA exchange in Mycobacterium, identifying genes controlled by cell cycle regulators in Saccharomyces cerevisiae, analyzing global regulation of transposition and transcription in Escherichia coil, as well as examining nucleic acid binding protein1target site interactions in novel ways. Furthermore, this array system will facilitate the development of improved bioinformatics methods for analysis of genomic expression data.

DESCRIPTION (provided by applicant): The long term goal of this proposal is to identify the gene(s) that influence complex behavior in the mouse. We have chosen habituation to an open field as our first behavior to be analyzed. Habituation is a form of memory whereby an animal must remember its surroundings (or a novel object) such that it can respond differently on its subsequent exposure to these surroundings. Our particular assay involves habituation to an open field. In this task, mice are placed in an open field chamber and their activity is recorded on three consecutive days. If their activity changes over the three days, the mice have "habituated" to the open field. This task is therefore considered a learning paradigm since it requires the mouse to remember its previous experience in the open field. We have recently obtained preliminary data indicating that a gene (or genes) influencing this trait resides on Chromosome (Chr.) 15 of the mouse. In this grant application, we would now like to clone this gene as well as study its characteristics and effects on behavior. Our specific aims are as follows: (1) to produce B6.D2 and D2.B6 speed congenics and subcongenics for regions of Chr. 15 in the mouse. These strains will be used to further map this habituation trait as well as provide useful strains for further behavioral studies; (2) to test Chr. 15 congenics and subcongenics for behavioral traits including habituation. A battery of tests will be used to determine if performance in the habituation task correlates with performances on other tasks. Alcohol and drug-related behaviors will also be screened; (3) to identify candidate genes for the genetic control of habituation. Candidate genes will be identified either by examining polymorphic differences between strains or by comparing differences in gene expression in target tissues; and (4) to confirm the identity of candidate genes by making transgenic mice. Three strategies will be considered--(1) BAC transgenesis with BACs containing candidate genes, (2) knockin mice containing regions of known polymorphisms in candidate genes or (3) knockout mice and complementation studies that will reveal the hemizygous influence of the candidate gene. These genetically engineered mice will be used to explore the in vivo effects of the detected polymorphisms. It is anticipated that the successful positional cloning of this gene will lead to new information on the pathways involved in the habituation process.

[unreadable] DESCRIPTION (provided by applicant): Genes that control complex phenotypic traits are often difficult to map and even more difficult to identify. However, the identification of these genes could have important implications in our understanding of certain disease processes and their etiologies. The specific aims of this proposal are designed to investigate the use of knockout/congenic strains of mice for the mapping and identification of genes involved in complex traits. Knockout/congenic strains of mice are ones where a target locus has been ablated by homologous recombination in embryonic stem (ES) cells and which have also been backcrossed at least 8 times to an inbred strain, usually B6. Thus, these strains differ from their inbred partner not only at the target locus (target of the ablation) but also at the flanking region derived from the ES cell genotype (usually of 129 origin). We would like to use these strains to investigate the polymorphic differences that exist between the B6 and 129 strains. We present preliminary data that these knockout/congenic strains may be useful in this regard. Our specific aims are: (1) to determine the phenotypic differences that exist between knockout/congenic strains and their inbred partners. Here, we will focus on mouse behavioral traits since they are difficult to map by more conventional techniques; (2) to identify and confirm that some of these differences between knockout/congenic strains and their inbred partners are due to residual passenger 129 genetic material linked to the target or differential locus. Subcongenic strains will be made for further refinement of the region and to be used as future tools for the eventual positional cloning of these genes. [unreadable] [unreadable]