ABSTRACT
Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.
Subject(s)
Beckwith-Wiedemann Syndrome/genetics , Chromosomes, Human, Pair 11 , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Carcinogenicity Tests , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/analysis , Gene Expression , Humans , Immunohistochemistry , Membrane Proteins , Mice , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , RNA, Messenger/genetics , Rats , Sequence Homology, Amino Acid , SwineABSTRACT
We identified in the EST database murine and human sequences similar, but not identical, to the members of the PC3/BTG/TOB family of cell cycle inhibitors. A conserved domain (aa 50-68) of the PC3 protein, the prototype member of the family, was used as a query. That domain has been shown by us to be necessary for the antiproliferative activity of PC3. A murine EST clone and a highly homologous human EST clone, containing the entire ORF, were chosen for sequencing. Comparison to databases and a phylogenetic tree analysis indicated that these EST clones are the mouse and human homologues of a gene that represents a novel member of the PC3/BTG/TOB family. This gene, named PC3B, is endowed with marked antiproliferative activity, being able to induce G(1) arrest, and is highly expressed in testis, in oocyte, and in preimplantation embryos. Analysis of its expression during murine development indicated a specific localization in the olfactory epithelium at midgestation, suggesting that PC3B might be involved in the differentiation of this neuronal structure. Human PC3B mapped to chromosome 11q23, as indicated by radiation hybrid analysis.
Subject(s)
Cell Cycle Proteins/genetics , Olfactory Mucosa/metabolism , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Base Sequence , Cell Cycle/genetics , Cell Cycle Proteins/chemistry , Cloning, Molecular , Conserved Sequence , Evolution, Molecular , Expressed Sequence Tags , Humans , Mice , Molecular Sequence Data , Multigene Family , Olfactory Mucosa/cytology , Open Reading Frames , Phylogeny , Proprotein Convertases , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The rat PC4 gene had been initially isolated as a nerve growth factor-inducible sequence in PC12 cells. Although its function remains unknown, recently it has been shown that PC4 is necessary to muscle differentiation and that it might have a role in signal transduction. We report the isolation of the human homolog of the rat PC4 gene, renamed here IFRD1 (interferon-related developmental regulator 1). Several human IFRD1 clones were identified by searching the EST database using the rat IFRD1 (PC4) cDNA as a query. An EST clone containing the entire ORF was chosen for sequencing. Human IFRD1 presented a predicted protein product of 453 amino acids, highly conserved (90.2% identity) compared to the rat IFRD1 (PC4) protein sequences. The mapping assignment of human IFRD1 to chromosome 7q22-q31 was retrieved from the UniGene database maintained at NCBI. A comparison of human IFRD1 (PC4) protein to databases revealed 47% identity to the protein encoded by the human gene SKMc15, originally isolated from a chromosome 3-specific library. Therefore, SKMc15 is a gene related to IFRD1, being the second member of a novel family. We analyzed their expression during murine development, and we found that mouse IFRD1 appears more expressed in specific differentiating structures at midgestation, while mouse SKMc15 is highly expressed soon after gastrulation and in the hepatic primordium, suggesting an involvement in early hematopoiesis.
