ABSTRACT
We carried out a screening of genes that are differentially expressed in normal mice and reeler mutants and are characterized by abnormal neuronal migration and neurite deployment due to defective Reelin signalling. A novel gene, provisionally named C61, was overexpressed in Reelin-deficient embryonic mouse brain RNA. C61 encodes a 3.7 kb mRNA that is brain specific and developmentally regulated, with predominant expression in differentiating neurons. The predicted protein is 664 amino acids long, and contains LAG1 and Ezrin/Radixin/Moesin-Myosin-Filament motifs, suggesting that it may function as an intracellular adaptor. From E14.5 to birth, C61 was highly expressed in all neuronal differentiation fields, with the highest signal in the telencephalic cortical plate and mitral cells in the olfactory bulb. When expressed as a GFP fusion protein in transfected non-neuronal cells and primary neurons, this protein localizes, respectively, to the nuclear membrane or axonal outgrowths, indicating a function in axonal traffic or signalling.
Subject(s)
Brain/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/physiology , Extracellular Matrix Proteins/physiology , Gene Expression Regulation, Developmental , Synapsins/metabolism , Amino Acid Motifs/physiology , Amino Acid Sequence , Animals , Animals, Newborn , Blotting, Northern/methods , Brain/embryology , Brain/growth & development , Caenorhabditis elegans , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line , Cloning, Molecular , Drosophila , Embryo, Mammalian , Embryo, Nonmammalian , Extracellular Matrix Proteins/deficiency , Extracellular Matrix Proteins/genetics , Green Fluorescent Proteins , Humans , Immunohistochemistry/methods , In Situ Hybridization/methods , Luminescent Proteins/metabolism , Membrane Proteins , Mice , Mice, Inbred BALB C , Mice, Neurologic Mutants , Microfilament Proteins , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins , Neurofibromin 2/genetics , Neurofibromin 2/metabolism , Neurons/metabolism , Organ Specificity , RNA, Messenger/biosynthesis , Reelin Protein , Reverse Transcriptase Polymerase Chain Reaction/methods , Serine Endopeptidases , Transfection , Tubulin/metabolism , ZebrafishABSTRACT
BACKGROUND: cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz. RESULTS: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development. CONCLUSION: We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.
