Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection.

Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We divided this cDNA expression library into multiple pools and transfected mammalian cells with the individual pools. Following transfection, we tested the expressed proteins for DNA-binding activity by agarose thin-layer electrophoretic gelshift assay. After we identified a single expression poolfor the presence of a DNA-binding protein, the corresponding cDNA pool was further divided into smaller aliquots. Then, the cDNA expression and gelshift clone selection was repeated until a single clone was isolated In contrast to traditional polyacrylamide gels, the agarose thin-layer is significantly faster and resolves larger DNA-protein complexes. This method can be widely used for the cDNA cloning of DNA- and RNA-binding proteins from various mammalian host cells.

Developmental changes in the inducibility of fos-like immunoreactivity in primary embryonic spinal cord cultures.

The immediate early gene (IEG) transcription factor c-fos coordinates changes in the pattern of long term gene expression and, therefore, it may be involved in mediating epigenetic control during neurodevelopment. We used pharmacological treatments mimicking various environmental and intracellular signals and assessed the inducibility of fos-like immunoreactivity (LIR) at various stages of neurodifferentiation in a primary embryonic spinal cord culture system by immunohistochemistry. Constitutive fos LIR exclusively found in neurons, was driven by the onset and extent of spontaneous electrical activity, as it was blockable by tetrodotoxin (TTX) at all developmental stages. Phorbol myristate 13 acetate (PMA) increased the number of fos-LIR cells equally effectively at all stages, but the predominant cellular localization of fos-LIR changed through ontogeny. The effect of veratridine, kainate and serum-derived factors in significantly inducing fos-LIR was restricted to the earliest developmental stage (4 days in vitro; DIV) investigated; whereas forskolin, the GABAA antagonist picrotoxin and NMDA failed to induce fos-LIR at this stage, but increased the number of fos-LIR neurons at later stages. Dihydropyridine agonists of the voltage-sensitive calcium channels (VSCC) raised the number of fos-LIR neurons and also prevented TTX-mediated down-regulation; whereas antagonists markedly reduced fos-LIR at all ages. Either type of NMDA antagonists (AP5 and MK801) and the GABAA agonist muscimol significantly reduced fos-LIR at all ages. These findings demonstrate that the inducibility of fos-LIR is substantially different in embryonic neurons than in adult ones and that inducibility by various first and second messengers is dependent on the development stage.

High conservation of upstream regulatory sequences on the human and mouse vasoactive intestinal peptide (VIP) genes.

The neuropeptide vasoactive intestinal peptide (VIP) gene is subject to complex transcriptional regulation resulting in expression of the encoded peptides in distinct subpopulations of neurons in most structures of the nervous system, and tissue-specific changes in expression in response to a variety of hormone and environmental factors. This diverse regulation allows the encoded peptides to carry out putative neurotransmitter, neuromodulator, trophic, neuroendocrine, and immune functions. Despite the potential significance of the processes governing its expression, only the human gene has been studied in any depth, and only a single regulatory element has been identified, a cAMP-responsive sequence less than 100 bp upstream from the transcriptional start site. Because tissue-specific patterns of VIP expression are remarkably well conserved between rodents and humans, we isolated the mouse VIP gene and compared 5' flanking sequences with that of the human gene to identify homologous regions which might be involved in regulation common to both species. Of significant interest is a 210 bp region located more than 1.1 kb upstream from the transcription start site that is 91% conserved between the two species. Of additional interest is a 34 bp perfect dCA.dTG repeat present only on the mouse gene which may be capable of forming Z-DNA.

Excitatory amino acid regulation of the enkephalin phenotype in mouse embryonic spinal cord cultures.

Expression of the preproenkephalin gene in developing spinal cord-dorsal root ganglia (SC-DRG) cultures was determined by Northern analysis following treatments with different agonists and antagonists of the glutamate receptor. Cultures (10-12 days old) were treated with various concentrations (10(-7)-10(-3) M) of N-methyl-D-aspartate (NMDA), quisqualate, kainic acid (KA), 2-amino-5-phosphonovaleric acid (APV) and 5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (MK801) either with or without blocking spontaneous electrical activity with 1 microM tetrodotoxin (TTX). In electrically active cultures, treatments with NMDA and KA increased preproenkephalin transcripts (mRNAppENK), showing maximum effects at 1 microM (4-fold and 2-fold, respectively), while treatments with quisqualate and MK801 caused concentration-dependent down-regulation in mRNAppENK. The most effective concentrations of NMDA (1 microM) and quisqualate (10 microM) altered mRNAppENK levels within 4 h of treatment and peaked after 24 h for NMDA and 48 h for quisqualate treatment. Co-treatment with APV completely blocked the NMDA-induced rise of mRNAppENK. During electrical blockade, none of the concentrations of NMDA tested showed any effect on enkephalin expression, neither could NMDA pre-treatment prevent the TTX-induced down-regulation of mRNAppENK. Our results indicate that the activity-dependent establishment of the enkephalin phenotype is modulated through the selective activation of the NMDA-glutamate receptor.