Further analysis of this cDNA revealed that, although several copies of an expressed Br-cadherin pseudogene are localized to the spinal muscular atrophy region, the full length, intact Br-cadherin gene is located on the opposite arm of chromosome 5, at 5p13C14 (17)

Further analysis of this cDNA revealed that, although several copies of an expressed Br-cadherin pseudogene are localized to the spinal muscular atrophy region, the full length, intact Br-cadherin gene is located on the opposite arm of chromosome 5, at 5p13C14 (17). during the first postnatal week, which corresponds temporally to the onset ENMD-2076 of synaptogenesis and dendrite outgrowth in the brain. This pattern of expression is consistent with a role for Br-cadherin in neuronal development, perhaps specifically with synaptogenesis. Cell adhesion molecules mediate contact-dependent processes that ENMD-2076 are essential requirements for cell migration and morphogenesis during development. The cadherins, a large family of cell surface molecules, are a well characterized group of transmembrane glycoproteins that ENMD-2076 function as cell adhesion molecules. Cadherins interact with each other via Ca2+-dependent, homophilic, and, less generally, heterophilic binding to other cadherin molecules (1, 2), as well as other cell adhesion molecules (3). In addition to having adhesive properties, cadherins are involved in cell signaling by activation of second messenger pathways; there is an accumulating body of evidence that shows this involvement (examined in refs. 4 and 5). Cadherins have a cannonic structure consisting of a long extracellular (EC) domain name of five repeats, located at the amino terminus of the protein (2, 6). Conserved motifs among different cadherins in the EC domain name include putative glycosylation and calcium-binding sites. A cell adhesion ENMD-2076 acknowledgement sequence, which is thought to facilitate binding, is present in the first EC repeat. After the repeats, the majority of cadherins have a single transmembrane domain name and a short and highly conserved cytoplasmic domain name that associates indirectly with the actin cytoskeleton via the catenin and -actinin proteins (7C9). Most cadherins are expressed both during embryonic development and in the mature organism (examined in refs. 4 and 9). The crucial role that cadherins play in neuronal development has been repeatedly exhibited. Neurulation, neuroepithelial development, and neurite outgrowth depend on the presence of cadherins (2, 6), and disturbance in their expression results in grossly abnormal development of the nervous system (10, 11). For example, injection of antibodies against N-cadherin into chicken embryos results in abnormalities of the neural tube and defective migration of the neural crest (12). Multiple cadherin genes are expressed in the nervous system (2, 5, 13, 14), but all are expressed in other tissues as well. Here we describe a new member of the cadherin family, Br-cadherin, whose protein is usually uniquely expressed in the brain. Previously, we cloned a partial cDNA of Br-cadherin as part of an effort to identify brain-derived transcripts from your spinal muscular atrophy region on human chromosome 5q13 (15, 16). Further analysis of this cDNA revealed that, although several copies of an expressed Br-cadherin pseudogene are localized to SCNN1A the spinal muscular atrophy region, the full length, intact Br-cadherin gene is located on the opposite arm of chromosome 5, at 5p13C14 (17). A partial sequence of the gene (designated as cadherin-12) was explained by Tanihara (18). The development course of Br-cadherin expression also is unique. Unlike other cadherins, Br-cadherin is usually detected only postnatally in the mouse, and its expression increases gradually during the first week of life to adult levels. The onset of expression in the mouse ENMD-2076 correlates with simultaneous increasing neurite outgrowth and synaptogenesis; thus, Br-cadherin is usually temporally and spatially well localized to play a role in a critical period in neurogenesis. MATERIALS AND METHODS DNA Sequencing and Intron/Exon Border Analysis. Genomic phages encompassing the human Br-cadherin locus were cloned as explained (17). Exon-containing restriction fragments from these phages were detected by hybridization to Br-cadherin cDNA. These fragments were subcloned into pBluescript II SK(+) plasmid vectors (Stratagene) and sequenced with primers based on the cDNA sequence. Sequencing was performed with an Applied Biosystems sequencer using DNA polymerase cycle sequencing, and acquired data were analyzed using sequencher software (Genecodes, Ann Arbor, MI). To determine intron/exon borders, the Br-cadherin cDNA sequence was compared with the genomic sequences by the Space function of Genetics Computer Group (Madison, WI) software. The presence of consensus splicing signals at points of sequence divergence was recognized by direct inspection. Intron Size Determination. Intron sizes were determined by PCR amplification of total human DNA or genomic phage DNA using cDNA primers situated in close proximity to intron/exon borders. For introns larger than 5 kb, TaKaRa Ex lover polymerase (Takara Shuzo, Kyoto) was used with extension occasions of 7C10 min at 72C for 30 cycles. PCR products were separated by electrophoresis on 0.4% agarose gels along with high molecular markers (GIBCO/BRL). Northern Blot Analysis. Northern blot analysis and 5-untranslated region (UTR) probe preparation were carried out as explained (17). Antibody Production. Antibodies for human Br-cadherin (anti-Br-cad-EC1) were generated against the peptide CPQYVGKLHSDLDKG from your amino terminus of the Br-cadherin protein (amino acids 72C85). The C residue, which is not present in Br-cadherin, was added to the amino terminus of the peptide as a linker for use in affinity purification. The peptide was synthesized, purified, coupled to keyhole limpet hemocyanin, and used.