ABSTRACT
Orthology is a widely used concept in comparative and evolutionary genomics. In addition to prokaryotic orthology, delineating eukaryotic orthology has provided insight into the evolution of higher organisms. Indeed, many eukaryotic ortholog databases have been established for this purpose. However, unlike prokaryotes, alternative splicing (AS) has hampered eukaryotic orthology assignments. Therefore, existing databases likely contain ambiguous eukaryotic ortholog relationships and possibly misclassify alternatively spliced protein isoforms as in-paralogs, which are duplicated genes that arise following speciation. Here, we propose a new approach for designating eukaryotic orthology using processed transcription units, and we present an orthology database prototype using the human and mouse genomes. Currently existing programs cover less than 69% of the human reference sequences when assigning human/mouse orthologs. In contrast, our method encompasses up to 80% of the human reference sequences. Moreover, the ortholog database presented herein is more than 92% consistent with the existing databases. In addition to managing AS, this approach is capable of identifying orthologs of embedded genes and fusion genes using syntenic evidence. In summary, this new approach is sensitive, specific and can generate a more comprehensive and accurate compilation of eukaryotic orthologs.
Subject(s)
Alternative Splicing , Databases, Genetic , Genomics , Sequence Homology, Nucleic Acid , Transcription, Genetic , Algorithms , Animals , Humans , Mice , Protein Isoforms/genetics , RNA, Messenger/chemistry , Sequence Homology, Amino Acid , SyntenyABSTRACT
To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34+ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.
Subject(s)
Cell Differentiation/physiology , Down-Regulation , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/physiology , Megakaryocytes/physiology , Nuclear Proteins/metabolism , Adult , Amino Acid Sequence , Animals , Antigens, CD34/metabolism , Biomarkers , Fetal Blood/cytology , Gene Expression Profiling , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Integrin beta3/metabolism , K562 Cells , Leukemia, Erythroblastic, Acute , Megakaryocytes/cytology , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Platelet Membrane Glycoprotein IIb/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Tetradecanoylphorbol Acetate/pharmacology , Tissue DistributionABSTRACT
DNA is a universal language encrypted with biological instruction for life. In higher organisms, the genetic information is preserved predominantly in an organized exon/intron structure. When a gene is expressed, the exons are spliced together to form the transcript for protein synthesis. We have developed a complexity reduction algorithm for sequence analysis (CRASA) that enables direct alignment of cDNA sequences to the genome. This method features a progressive data structure in hierarchical orders to facilitate a fast and efficient search mechanism. CRASA implementation was tested with already annotated genomic sequences in two benchmark data sets and compared with 15 annotation programs (10 ab initio and 5 homology-based approaches) against the EST database. By the use of layered noise filters, the complexity of CRASA-matched data was reduced exponentially. The results from the benchmark tests showed that CRASA annotation excelled in both the sensitivity and specificity categories. When CRASA was applied to the analysis of human Chromosomes 21 and 22, an additional 83 potential genes were identified. With its large-scale processing capability, CRASA can be used as a robust tool for genome annotation with high accuracy by matching the EST sequences precisely to the genomic sequences.
