RESUMEN
The thrombospondins (TSP-1, -2, and -3) comprise a family of proteins that are homologous at the carboxy terminus but have unique sequences at the amino terminus that might be correlated with the regulation of cell behavior. To investigate the expression of TSP-1, -2, and -3 in endothelial cells, we examined developing murine blood vessels and human atherosclerotic plaques by in situ hybridization. The expression of TSP-1 was also characterized in cultured bovine aortic endothelial cells. Expression of TSP-2 was seen in the dorsal aorta as early as embryonic day 10; TSP-1 was not detected in endothelial cells until later stages, and TSP-3 was not apparent in the vasculature. In atherosclerotic specimens, TSP-1 mRNA was detected in many intraplaque microvessels and in the endothelium lining the atheromatous plaque; TSP-2 was absent from these regions. Cultured bovine aortic endothelial cells did not transcribe TSP-2 mRNA at detectable levels. There were high steady-state levels of TSP-1 mRNA in subconfluent bovine aortic endothelial cells before confluence and at the wound edge after injury of the cell monolayer, with maximal expression of TSP-1 in cultures at a time during which approximately 35% of the cells were in S phase. As the majority of these cells subsequently undergo mitosis, these data are consistent with TSP-1 as an inhibitor of endothelial cell proliferation that functions in G1. These results support the conclusion that, despite sequence homology, the TSPs have distinct functions in vascular biology.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Bovinos , Moléculas de Adhesión Celular/metabolismo , Recuento de Células , División Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/patología , Femenino , Humanos , Ratones , TrombospondinasRESUMEN
We report the remainder of the cDNA sequence for human thrombospondin 2 (hTSP2) including the 5' and 3' untranslated regions. The homology between the two human thrombospondins is highest at the carboxyl-terminal end. The 5' untranslated region of hTSP2 is a GC-rich, 239-bp segment that encodes an in-frame methionine residue embedded in a Kozak consensus sequence. The 3' untranslated region is AT-rich and contains nucleotide sequences implicated in the regulation of mRNA stability. It also contains an 89-bp AT-rich segment with 92% of its nucleotides identical to the same region of the mouse TSP2 3' untranslated region, which suggests an important functional or structural property associated with this region.
Asunto(s)
Genes , Glicoproteínas de Membrana/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia , TrombospondinasRESUMEN
A novel form of human thrombospondin was identified during the screening of a human fibroblast cDNA library. We report the cDNA sequence for 1.8 kb of the 3' end of the cDNA, plus an additional 937 bp of 3'-untranslated sequence. The translated sequence reveals a high degree of similarity to thrombospondin I. The homology ranges from 56 to 80% for different regions within the two proteins. The repeating segments of amino acid sequence identified in thrombospondin I were found to be conserved in thrombospondin II. The new form of thrombospondin hybridizes to a 7.5-kb message by Northern analysis. The THBS2 gene is located at the distal long arm of chromosome 6 at 6q27. The gene is transcribed in fibroblasts, smooth muscle cells, and an osteosarcoma cell line, at levels somewhat lower than that of thrombospondin I. Umbilical vein endothelial cells do not transcribe thrombospondin II under the conditions of this study. These findings suggest that previous studies of thrombospondin function need to be reassessed to identify the functions specific to each molecule.
Asunto(s)
Cromosomas Humanos Par 6 , ADN/genética , Familia de Multigenes , Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células Cultivadas , Bandeo Cromosómico , Mapeo Cromosómico , Cricetinae , ADN/aislamiento & purificación , Fibroblastos/fisiología , Expresión Génica , Biblioteca de Genes , Humanos , Células Híbridas/fisiología , Recién Nacido , Lectinas/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Fenómenos Fisiológicos de la Piel , TrombospondinasRESUMEN
Evidence is presented that a sporulation-essential sigma factor of Bacillus subtilis, sigma 29, is synthesized as an inactive precursor (P31) and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the P31 amino terminus. A pulse-chase experiment demonstrated that sigma 29 was derived from a preexisting protein, with appearance of radioactively labeled sigma 29 paralleling the disappearance of labeled P31. The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B. subtilis strain carrying a mutation in a locus (spoIIE) required for sigma 29, but not P31, synthesis. Microsequencing of sigma 29 protein revealed that its amino terminus originates at amino acid 30 of the P31 amino acid sequence. In order to test whether a proteolytic event alone could activate P31 to a protein with sigma 29-like properties, a fusion protein (P31*) containing most of P31 was overproduced in Escherichia coli and converted in vitro into a protein with the electrophoretic mobility of sigma 29 by limited treatment with Staphylococcus aureus V8 protease. Protease-treated P31*, but not untreated P31*, was capable of directing B. subtilis core RNA polymerase to specifically initiate RNA synthesis at a sigma 29-recognized promoter in vitro.