Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


Transcript profiling of hematopoietic cell development and activation

Yamaga, S.1, Yeramilli, S.5, Lian, Z.1, Prashar, Y.5, Lee, H.1, Berliner, N.2, Liu, Y-C.1, Goguen, J.3, Newburger, P.4, and Weissman, S.1

1Yale University School of Medicine, Department of Genetics, New Haven, Connecticut, USA
2Yale University School of Medicine, Department of Internal Medicine, New Haven, Connecticut, USA
3University of Massachusetts Medical Center, Department of Molecular Genetics and Microbiology, Worcester, Massachusetts, USA
4University of Massachusetts Medical Center, Pediatrics, Worcester, MA, USA
5Gene Logic Inc., Gaithersburg, Maryland, USA

The hematopoietic system is a favorable mammalian system for studying changes in gene expression during differentiation and activation. At least twelve lineages of cells are derived from a common precursor. Several of these cell types can be isolated in convenient numbers and purity for analysis. Model cellular systems exist for certain stages of in-vitro differentiation. Cells from specific types of hematopoietic neoplasia can also be obtained in pure form without extensive manipulation. We have relied principally, but not solely, on 3' end restriction fragment gel display methods to profile gene expression in several stages of differentiation of myeloid cells and, to a lesser extent, in cells of several other lineages. Neutrophil activation has been studied intensively. Combinatorial use of genes in different lineages is striking. For example, the Lander group (Cell 97:227, 1999) has studied the effects of activation of murine 3T3 cells by signaling through the cytoplasmic domain of the platelet derived growth factor receptor. They found 66 genes that were actively up-regulated within 4 hours after the onset of stimulation. Of these genes, at least 45% are actively up-regulated in mature neutrophils within 2 hours after addition of non-pathogenic bacteria, although the neutrophils are post-mitotic, highly specialized cells. The overall short term response of the neutrophils, however, involved changes in the levels of more than 600 mRNAs, achieved by changes in transcription but also by both up- and down-regulation of the stability of specific mRNAs. Biologic effects that are at least partially interpretable include the development of new autocrine loops, complex anti-apoptotic changes, and changes favoring cell survival and increased membrane trafficking. In the process of this work certain complications became apparent that would probably be shared by all approaches to cDNA profiling that involve an oligo-dT priming step. These affect only a minor subset of the products. In certain cases investigation is needed to determine the possible biologic significance of particular truncated mRNAs.


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