Microchip arrays of regulatory genes

Abstract

Microarray technology is a fast-growing field of biomedical research, aiming to investigate changes in molecular features of hundreds of genes. The multiple parallel processing of information generated from matrices of huge numbers of loci on a solid substrate has allowed the gathering of gene signatures defining specific biological states. A new approach has been developed to facilitate this process wherein genes of the same regulatory modality are selected. The transcriptional regulation of these genes is related to the same control element, the E-box, defined by the sequence CACGTG. PCR products of selected regions of all known genes either binding to this sequence or whose expression is dependent on this binding, as well as genes interacting with E-box-binding genes and control genes, are arrayed on a nylon membrane or other appropriate microchip susbstrate, which is then used as an E-box-specific microarray. The transcriptionally regulated profile of E-box-related genes specific to a given cultured cell sample is then determined by unique labeled cDNAs probes produced from RNAs isolated from the culture of interest.

1 . An array of genes each comprising an E-box regulatory sequence in its promoter, interacting with a gene binding to the E-box regulatory sequence or whose expression is dependent on this binding.  
     
     
         2 . The array of  claim 1  wherein the genes further comprise sequences encoding proteins associated with a particular state, disease or disorder.  
     
     
         3 . The array of  claim 2  wherein the state is age.  
     
     
         4 . The array of  claim 3  wherein the genes are isolated from a young animal.  
     
     
         5 . The array of  claim 3  wherein the genes are isolated from an old animal.  
     
     
         6 . The array of  claim 2  further comprising housekeeping genes whose expression does not change significantly as the state changes.  
     
     
         7 . The array of  claim 6  comprising at least nine housekeeping genes.  
     
     
         8 . The array of  claim 6  wherein the state is age and the expression of the housekeeping genes does not change as the animals age.  
     
     
         9 . The array of  claim 8  wherein the housekeeping genes are selected from the group consisting of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, hypoxanthine phosphoribosyltransferase I (Lesh-Nyhan syndrome), Major histocompatibility complex, class I, C, Ubiquitin C, Glyceraldehyde-3-phosphate dehydrogenase, Human mRNA fragment encoding cytoplasmic actin, 60S Ribosomal protein L13A, and Aldolase C.  
     
     
         10 . The array of  claim 1  wherein the genes are present in nanomolar quantities.  
     
     
         11 . A set of primers for use in detecting changes in expression of genes comprising an E-box regulatory sequence in its promoter or interacting with a gene binding to the E-box regulatory sequence, having a length between 480 and 700 base pairs length and a melting point between 75 and 85° C., wherein the primers include non-consensus sequence with protein coding sequence so that there is no detectable hybridization between homologous genes.  
     
     
         12 . The set of primers of  claim 11  where there is no hybridization between homologous genes.  
     
     
         13 . The set of primers of  claim 11  wherein the primers do not hybridize to homologous genes having the same degree of homology and c-myc and c-myc associated genes.  
     
     
         14 . The set of primers of  claim 11  comprising a label.  
     
     
         15 . The set of primers of  claim 14  wherein the label is selected from the group consisting of digoxigenine label, radiolabels and fluorescent labels.  
     
     
         16 . A kit for detecting changes in expression of genes which is associated with a particular state, disease or disorder comprising 
 a) an array of genes each comprising an E-box regulatory sequence in its promoter or interacting with a gene binding to the E-box regulatory sequence and one or more housekeeping genes;    b) a set of E-box primers for use in detecting changes in expression of the genes in the array; and    c) means for detecting hybridization between the primers and the regulatory sequences.    
     
     
         17 . The kit of  claim 16  wherein the array of genes is prepared from cells or tissues of an animal characterized by a particular state, disease or disorder.  
     
     
         18 . The kit of  claim 17  wherein the animal is selected based on age or a disease or disorder associated with cancer, the neuronal system, the musculoskeletal system, or cardiovascular system.  
     
     
         19 . The kit of  claim 17  further comprising means for quantitating the amount of expression.  
     
     
         20 . The kit of  claim 18  wherein the animal or cells from which the genes are derived have been treated with one or more compounds or dosage regimes to screen for an effect of the compound or dosage regime on the state, disease or disorder.  
     
     
         21 - 29 . (canceled)