Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action.

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals.

Assessing gene expression variation in normal human tissues using GeneTag, a novel, global, sensitive profiling method.

GeneTag is a novel expression profiling method that allows the visualization, quantification and identification of expressed genes-whether known or novel-in any species, tissue or cell type, independent of knowledge of the underlying sequence. Here we describe the application of this method to determine variation of gene expression in individual human liver samples and the identification of tissue-specific genes by comparing expression patterns across several human organs. Expression data are stored in a database for future reference and data analysis relies on proprietary software, which allows complex comparisons to be performed.

Identification of ubiquitin ligases required for skeletal muscle atrophy.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models.

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.

General method for HPLC purification and sequencing of selected dsDNA gene fragments from complex PCRs generated during gene expression profiling.

Gene expression profiling using an AFLP-based technique generates a large number of gene fragments that require identification by sequencing. The DNA fragments vary in length from about 50-500 bp. Ion-pair reversed-phase HPLC can be used to purify selected double-stranded DNA fragments that represent differentially expressed genes.

A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).

Currently two mutations in the HFE gene are known to be associated with the manifestation of the autosomal recessive disorder hereditary hemochromatosis (HH). A single-base mutation resulting in Cys282Tyr appears to have a causative role in the development of the disease, and a point mutation resulting in His63Asp may also be involved. Recent observations with a fully automated capillary electrophoresis (CE) system (ABI Prism 310) suggested that this instrument could be used for the precise identification of known mutations based on single-strand conformation polymorphism (SSCP).

Hematopoietic defects in mice lacking the sialomucin CD34.

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown.

Selective modulation of the expression of L-selectin ligands by an immune response.

Background: The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L-selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells.

The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development.

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver.

Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells.

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor.

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin.

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level.

HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF.

The coordinate expression of genes during development and differentiation is thought to be accomplished by common transcription factors operating on the promoters of families of coexpressed genes. HNF-1 is a transcriptional factor involved in the expression of genes in the liver and was originally defined as playing a major role in coordinating the expression of the linked fibrinogen genes. We have isolated cDNA clones for HNF-1 using oligonucleotides prepared to the sequence of the purified protein.

Purified hepatocyte nuclear factor 1 interacts with a family of hepatocyte-specific promoters.

During development cell types arise through the activation or repression of classes of specific genes. One hypothesis is that this phenomenon is realized by tissue-specific factors playing a role at the transcription level. Recently we have described a liver-specific nuclear protein, hepatocyte nuclear factor 1, that appears to be involved in the transcription of the fibrinogen and alpha 1-antitrypsin genes.

A variant nuclear protein in dedifferentiated hepatoma cells binds to the same functional sequences in the beta fibrinogen gene promoter as HNF-1.

Normal liver and differentiated hepatoma cell lines contain a nuclear factor, HNF-1, which binds functional sequences within the promoters of the alpha and beta chains of fibrinogen and alpha 1-antitrypsin. In UV cross-linking studies we find that HNF-1 has an apparent mol. wt of 92 kd in differentiated hepatocytes.