RESUMO
Neurocan and brevican are related chondroitin sulfate proteoglycans which are mainly expressed in the central nervous system. Neurocan and the secreted brevican variant are composed of globular N-terminal hyaluronan binding domains, central O-linked oligosaccharide attachment regions, and globular C-terminal domains. Interaction studies of mouse brain proteoglycans revealed that neurocan, but not brevican, was retained on a heparin affinity matrix. Also a recombinantly produced C-terminal fragment of neurocan, expressed by HEK 293 cells, was retained by the heparin affinity matrix. The substitution of this fragment with a chondroitin sulfate chain did not inhibit binding to the heparin affinity matrix at physiological NaCl concentrations, but decreased the NaCl concentration necessary for elution. Two potential consequences of the heparin binding ability of neurocan are an enforcement of the interaction with other heparin binding molecules and a directed secretion by polarized cells.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Heparina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Western Blotting , Encéfalo , Brevicam , Linhagem Celular , Polaridade Celular , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Cromatografia de Afinidade , Cães , Glicosilação , Heparina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Lectinas Tipo C , Camundongos , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Neurocam , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tenascina/química , Tenascina/metabolismo , TransfecçãoRESUMO
Cosmid clones containing the mouse neurocan gene were isolated from a genomic library using rat neurocan cDNA fragments as probe. The murine gene has a size of approximately 25 kb and contains the coding sequence for the mRNA on 15 exons. The exon-intron structure reflected the structural organization of neurocan, which is a multidomain protein belonging to the aggrecan/versican proteoglycan family. All introns between conserved modular protein domains are phase I introns. Primer extension experiments indicate a transcriptional start point 28 bases downstream of a consensus TATA sequence. Further analysis of 1 kb of 5' flanking sequence revealed in addition to AP1, AP2, and SP1 consensus binding sites multiple E-box elements and a glucocorticoid responsive element. Single-strand conformation polymorphism was used to map neurocan to chromosome 8 between the microsatellite markers D8Mit29 and D8Mit78. Among mouse mutants that have been mapped around this region are the three allelic neurological diseases tottering, leaner, and rolling. The multidomain structure and the preferential expression of neurocan in the brain suggest a potential involvement in these diseases.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas do Tecido Nervoso/genética , Animais , Sequência de Bases , Sistema Nervoso Central/crescimento & desenvolvimento , Mapeamento Cromossômico , DNA , Éxons , Íntrons , Lectinas Tipo C , Camundongos , Dados de Sequência Molecular , Neurocam , Ratos , Transcrição GênicaRESUMO
An improved purification procedure for Thermotoga maritima RNA-polymerase holoenzyme was developed. The enzyme is highly active with poly dAT or T7 phage DNA as template. DNA gyrase was found to be a side product of this RNA-polymerase purification. The genes for the large subunits beta and beta' of RNA-polymerase were cloned and sequenced. The phylogenetic position of T.maritima within the bacterial domain was determined by various methods. It is the lowest bacterial offspring but slightly higher than the chloroplasts.