RESUMO
A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.
Assuntos
Quimosina/genética , Clonagem Molecular , DNA/metabolismo , Precursores Enzimáticos/genética , Escherichia coli/genética , Animais , Sequência de Bases , Bovinos , Quimosina/metabolismo , Enzimas de Restrição do DNA , Precursores Enzimáticos/metabolismo , Plasmídeos , RNA Mensageiro/genéticaRESUMO
DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.
Assuntos
Quimosina/genética , Clonagem Molecular , DNA , Precursores Enzimáticos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Enzimas de Restrição do DNA , Biossíntese de Proteínas , RNA Mensageiro/genética , Estômago/enzimologiaRESUMO
Two dodecadeoxynucleotides of defined sequence have been synthesised by phosphotriester methodology. They can be polymerised to give a double stranded DNA which codes, when read in the correct phase, for the repeating dipeptide poly(aspartyl-phenylalanine). This polymeric DNA has been cloned in E. coli K12 using as vector a plasmid having a controllable bacterial promoter upstream of the insertion site. Clones containing genes coding for up to 150 repeats of (aspartyl-phenylalanine) have been isolated and characterised. The polymeric inserts appear to be stable over many generations and are expressed in E. coli under the control of the bacterial promoter, to give a polymer of phenylalanine and aspartic acid which may be broken down enzymically to yield aspartyl-phenylalanine.
Assuntos
DNA Recombinante , Escherichia coli/metabolismo , Genes , Biossíntese Peptídica , Peptídeos , Sequência de Aminoácidos , Clonagem Molecular , Plasmídeos , Biossíntese de Proteínas , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaAssuntos
DNA/genética , Genes Virais , Genes , Transcrição Gênica , Animais , Sequência de Bases , Evolução Biológica , Enzimas de Restrição do DNA , Código Genético , Globinas/genética , Humanos , Imunoglobulinas/genética , Hibridização de Ácido Nucleico , Ovalbumina/genética , RNA Mensageiro/genéticaRESUMO
Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.
Assuntos
DNA/genética , Genes , Ovalbumina/genética , RNA Mensageiro/genética , Animais , Galinhas/genética , Enzimas de Restrição do DNA , Fígado , Peso Molecular , Hibridização de Ácido Nucleico , Oviductos , PlasmídeosRESUMO
We present evidence that the poly(A) sequence at the 3' end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system. Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact. It is shown that the stability of the mRNA in a cell free system is not appreciably affected by this procedure. Measurements of the size of ovalbumin-synthesizing polysomes, rate of peptide elongation, and number of rounds of translation per messenger show a generally reduced efficiency for deadenylated mRNA compared to native mRNA. No comparable difference was observed in experiments with a wheat germ cell-free system, which gives few rounds of translation per mRNA. This indicates that the effect results from a lowering of the efficiency of reinitiation on deadenylated mRNA.
