RESUMO
We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Oócitos/metabolismo , Espermatozoides/metabolismo , Animais , Sequência de Bases , Extratos Celulares , DNA/metabolismo , Replicação do DNA , Immunoblotting , Masculino , Dados de Sequência Molecular , Nucleossomos/metabolismo , Fosforilação , Xenopus laevisRESUMO
Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter. When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites. Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon. We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts.
Assuntos
Códon/genética , Genes Virais , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Sequência de Bases , Sistema Livre de Células , DNA Viral/genética , Vetores Genéticos , Vírus da Influenza A/genética , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Coelhos , Mapeamento por Restrição , Reticulócitos/metabolismo , Fagos T/genéticaRESUMO
As a test of the fidelity of the rabbit reticulocyte lysate system, we have examined the products of translation of various different influenza virus mRNAs, produced by in vitro transcription. A common finding with all mRNA species was that the ratio of full-length translation product to incomplete products decreased with increasing mRNA concentration. These short products are a mixture of (i) polypeptides initiated at the authentic initiation site but terminated prematurely, and (ii) polypeptides initiated at internal sites and terminated at the correct site. Analysis of mRNA stability during the translation assay showed very little degradation, quite insufficient to be the principle cause of incomplete product synthesis. Investigation of the influence of various parameters on the ratio of full-length to incomplete products leads to the conclusion that a high fidelity of translation can be obtained provided certain precautions are followed: the use of capped, rather than uncapped, mRNAs at low concentrations, with KCl concentrations about 20 mM above the level that gives maximum incorporation.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/genética , Reticulócitos/fisiologia , Animais , Sistema Livre de Células , Técnicas In Vitro , Células L/fisiologia , Peso Molecular , Orthomyxoviridae/genética , Iniciação Traducional da Cadeia Peptídica , Cloreto de Potássio/farmacologia , Testes de Precipitina , Capuzes de RNA , CoelhosRESUMO
A cDNA clone of the influenza virus NS (non-structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in-frame AUG codons 57 nucleotide residues apart. Each initiation site was in either a preferred context (...AUAAUGG...) or a less favourable context (...UUUAUGG...) and the four possible permutations were constructed. When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons were observed. At low RNA concentrations the frequency of initiation at the 5'-proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model. However, a completely unexpected finding was that the ratio of initiation at the first AUG codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved. Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out-titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5'-proximal AUG codon. The effect of supplementing the system with purified initiation factors was examined. Only eIF-2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF-3, 4A, 4B, 4C + 4D, 4F and 5 were inactive.