ABSTRACT
A full-length cDNA clone of a previously unidentified serine protease, myelencephalon-specific protease (MSP), has been isolated by using a PCR cloning strategy and has been shown to be expressed in a nervous system and spinal cord-specific pattern. Sequence analysis demonstrated that MSP is most similar in sequence to neuropsin, trypsin, and tissue kallikrein and is predicted to have trypsin-like substrate specificity. MSP mRNA was found to be approximately 10-fold greater in the CNS of the rat and human, as compared with most peripheral tissues, and within the CNS was found to be highest by a factor of four in the medulla oblongata and spinal cord. Levels of mRNA encoding tissue plasminogen activator (tPA) also were elevated in the spinal cord but were more widespread in peripheral tissues as compared with MSP. In the adult rat lumbosacral spinal cord, in situ localization of MSP mRNA demonstrated 2-fold higher levels in the white, as compared with the gray, matter. MSP mRNA expression was shown to increase 3-fold in the white matter and 1.5-fold in the gray laminae at 72 hr after intraperitoneal injection of the AMPA/kainate glutamate receptor-specific agonist, kainic acid (KA). MSP mRNA remained elevated in the ventral gray matter, including expression associated with the motor neurons of lamina IX, at 7 d after the initial excitotoxic insult. Together, these observations indicate that MSP is in a position to play a fundamental role in normal homeostasis and in the response of the spinal cord to injury.
Subject(s)
Gene Expression Regulation/drug effects , Kainic Acid/toxicity , Nerve Tissue Proteins/biosynthesis , Neurotoxins/toxicity , Serine Endopeptidases/biosynthesis , Spinal Cord/drug effects , Amino Acid Sequence , Animals , Base Sequence , Brain Chemistry , DNA, Complementary/genetics , Genes , Humans , In Situ Hybridization , Kainic Acid/pharmacology , Male , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nervous System/chemistry , Neurotoxins/pharmacology , Organ Specificity , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Sequence Alignment , Sequence Homology, Amino Acid , Serine Endopeptidases/genetics , Spinal Cord/metabolism , Substrate SpecificityABSTRACT
To examine the content of the 5' flanking region of the mouse BDNF gene a mouse library was screened using oligonucleotides corresponding to the rat exon I untranslated region. A 6-kb genomic fragment containing exons I and II and flanking regions was isolated and sequenced. The structure of the 5' end of the mouse gene is similar to that of rat, exons I and II are 2 small untranslated regions clustered within 500 bp of each other at the 5' end of the gene. The nucleotide sequence homology between rat and mouse is 93%. Analysis for transcription factor-binding sites show a predominance of AP1 and C/EBP elements which are conserved between the 2 species. Deleted fragments of the 5' flanking region of exons I and II were fused to the luciferase reporter gene and transcriptional activity was analyzed by transient expression in primary cortico-hippocampal cultures. We found that a fragment of 266 bp from exon I transcription start is sufficient for promoter activity in basal conditions. Following experimental stimulation by treatment with kainic acid, we determined that regulatory elements responsive to kainic acid are located within 989 bp of the transcriptional start of exon I.
Subject(s)
Brain-Derived Neurotrophic Factor/genetics , Cerebral Cortex/metabolism , Hippocampus/metabolism , Neurons/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Brain-Derived Neurotrophic Factor/biosynthesis , Cells, Cultured , Cloning, Molecular , Exons , Fetus , Gene Library , Luciferases/biosynthesis , Mice , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Transcription, Genetic , TransfectionABSTRACT
We have used the polymerase chain reaction (PCR) to amplify and clone coding sequences of the mature region of brain-derived neurotrophic factor (BDNF) from monkey, rat, chicken and Xenopus genomic DNA. Consistent with previous reports, the predicted amino acid sequences obtained in this manner from monkey and rat were identical to other mammalian BDNF sequences. The chicken and Xenopus BDNF sequences are also highly conserved, but contain 7 and 8 amino acid substitutions, respectively, compared to mammalian BDNF. Comparison of these sequences with the homologous NGF and NT3 coding regions provides further insight into amino acid residues that may be responsible for the different receptor specificities of these factors.
