Isolation of cDNA encoding for secreted or membranal proteins

Details Isolation of cDNA encoding for secreted or membranal proteins Isolation of cDNA encoding for secreted or membranal proteins

2000-03-24

2002-06-11

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007200, C435S091100, C435S287200

Reexamination Certificate

active

06403316

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the identification of cDNAs and genes encoding secreted or membranal coding mRNAs. More specifically, the present invention relates to a method of identifying clones encoding for membranal and secreted proteins by deriving probes from template RNA extracted from membrane-bound polysomes and free polysomes and performing microarray-based comparison of the relative abundance of different RNA species. Analysis of the results of such comparison and identification thereby of clones encoding for membranal or secreted proteins provides a valuable tool which may be used together with other gene discovery tools, and which in itself enables identification of likely targets for drug development.
2. Description of Related Art
The discovery of novel genes and the elucidation of their role in normal and pathological processes is a major goal of biomedical research. Gene expression profiling with cDNA microarrays is currently widely used to obtain broad information on expression patterns of thousands of genes in conjunction with various biological conditions (Spellman et al., 1998; lyer et al., 1999; Feng et al., 1999; Wang et al., 1999; Whitney et al., 1999). Schena et al. developed a high capacity system to monitor the expression of many genes in parallel utilizing microarrays. The microarrays are prepared by high speed robotic printing of cDNAs on glass providing quantitative expression measurements of the corresponding genes (Schena et al., 1995). Differential expression measurements of genes are made by means of simultaneous, two color fluorescence hybridization. Other methods that may be used for differential expression measurement are, for example, Affymetrix, oligochip, SAGE, differential display and its variants and subtractive hybridization.
Powerful bioinformatics tools have been developed in order to classify and identify interesting genes according to their gene expression profiles (Eisen et al,m 1998; Bassett et al., 1999) and associate them with a condition of interest. The identification and/or isolation of genes whose expression differs between two cell or tissue types, or between cells or tissues exposed to stress conditions, chemical compounds or pathogens, is critical to the understanding of mechanisms underlying various physiological conditions, disorders, or diseases. However, out of the many clones that are identified based on interesting expression patterns, only those representing known genes can be considered to be promising drugs or drug-targets. A significant portion of the differentially expressed, arrayed clones, are unknown ESTs frequently derived from untranslated cDNA regions or containing no informative structural protein patterns. Thus, no clues exist as to the potential function or sub-cellular localization of a large group of clones.
Since membranal and secreted proteins are both accessible and critical for transduction of numerous intra- and intercellular signals, they are generally viewed as preferred targets for pharmacological use and intervention. Therefore, the a priori classification of arrayed unknown gene sequences into those that potentially code for secreted and membranal proteins is of great value for the optimization of a high-throughput process of identifying potential drug targets. Furthermore, it would be useful to further identify additional genes which express membranal or secreted proteins that are differentially expressed in different, cellular situations is of the utmost importance in designing therapeutic or diagnostic tools.
SUMMARY OF THE INVENTION
The present invention provides a method of identifying clones which encode membranal and secreted proteins by preparing cell fractionations, preparing cDNA probes from template RNA derived from membrane-bound polysomes and free-polysomes, performing a microarray-based comparison of the relative abundance of different RNA species, analyzing the results and thereby identifying genes encoding for membranal and secreted proteins. Since membranal and secreted proteins are generally viewed as preferred targets for pharmacological intervention, the present invention thus provides a method of identifying likely targets for drug development. The present invention further provides a method of augmenting microarray analysis by utilizing RNA extracted from specific subcellular-compartments as templates for DNA probes.


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Mechler, B.M. “Isolation of Messenger RNA from Membrane-Bound Polysomes” Methods in Enzymology, 1987, 152:241-248.

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