RESUMO
The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.
Assuntos
Proteínas de Transporte/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação ao GTP/genética , Células de Schwann/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Prenilação de Proteína , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.
Assuntos
Heparitina Sulfato/genética , Glicoproteínas de Membrana/genética , Proteoglicanas/genética , Envelhecimento , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Northern Blotting , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Clonagem Molecular/métodos , DNA/genética , DNA/isolamento & purificação , Embrião de Mamíferos , Imunofluorescência , Biblioteca Gênica , Proteoglicanas de Heparan Sulfato , Humanos , Fígado/crescimento & desenvolvimento , Fígado/fisiologia , Glicoproteínas de Membrana/análise , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteoglicanas/análise , RNA Mensageiro/genética , Ratos , Células de Schwann/fisiologia , Homologia de Sequência do Ácido Nucleico , Sindecanas , Transcrição GênicaRESUMO
The mechanism(s) by which rRNA accumulates during the growth of cardiac myocytes was investigated. The rates of rDNA transcription were measured in contracting myocytes and compared with nonbeating myocytes depolarized with 50 mM KCl. After 3 days of contraction the absolute rate of rDNA transcription was accelerated by 2-fold as measured by incorporation of [3H]UTP into the external transcribed spacer of preribosomal RNA. Corresponding increases in transcription were observed in isolated nuclei of contracting myocytes as measured by either hybridization of run-on transcripts of preribosomal RNA or activity of RNA polymerase I. The extent to which transcription was stimulated in contracting myocytes accounted for the previously observed acceleration of rRNA synthesis rates. The steady-state levels of preribosomal RNA relative to rRNA were unchanged in contracting myocytes, but the total amount of preribosomal RNA was 1.3-fold greater as a result of increased rRNA content. The increase of preribosomal RNA in proportion to rRNA in contracting myocytes demonstrated that the rate of preribosomal RNA processing was unchanged and that rRNA synthesis is regulated by an accelerated rate of rDNA transcription.