Helin Dong

Genome-wide expression monitoring in Saccharomyces cerevisiae

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

Transcriptional regulation and function during the human cell cycle

We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle–dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle–regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

Myoseverin, a microtubule-binding molecule with novel cellular effects

A new microtubule-binding molecule, myoseverin, was identified from a library of 2,6,9-trisubstituted purines in a morphological differentiation screen. Myoseverin induces the reversible fission of multinucleated myotubes into mononucleated fragments. Myotube fission promotes DNA synthesis and cell proliferation after removal of the compound and transfer of the cells to fresh growth medium. Transcriptional profiling and biochemical analysis indicate that myoseverin alone does not reverse the biochemical differentiation process. Instead, myoseverin affects the expression of a variety of growth factor, immunomodulatory, extracellular matrix-remodeling, and stress response genes, consistent with the activation of pathways involved in wound healing and tissue regeneration.

Deciphering Molecular Circuitry Using High-Density DNA Arrays

The immense amount of sequence data available from expressed sequence tag (EST) databases (1,2), together with the development of technologies for the highly parallel analysis of gene expression (3–5) have created the opportunity to interrogate biochemical pathways and gene function on an unprecedented scale. We describe here a set of high-density DNA arrays containing oligonucleotides complementary to more than 6,500 human EST’s. These arrays were used to generate normal and breast cancer specific gene expression profiles. More than 1,500 expressed genes were detected in both cell types examined with 85% of all gene expression observed in the range of 1–50 copies per cell. Over 300 genes demonstrated significantly different levels of expression between normal and transformed breast cells. Increased mRNA levels were observed for the Her2/neu oncogene and genes involved in its signal transduction pathway, including Grb-7, Ras, Raf, Mek and ERK. In addition, a simple categorization of the expression changes revealed patterns characteristic of loss of wild-type p53 function. Genotyping of the p53 locus using a DNA re-sequencing array revealed inactivating mutations in the p53 DNA binding domain and loss of heterozygosity. These data demonstrate a general array-hybridization-based approach to deciphering biochemical pathways and generating testable hypotheses concerning the mechanisms of cell growth and differentiation.