RESUMO
From a colony bank of HindIII-generated yeast DNA fragments we have isolated a number of recombinant DNAs carrying genes for ribosomal proteins (e.g., S10, S16A, S20, S24, S31, S33, L16, L25 and L34) of the yeast Saccharomyces carlsbergensis. By electron microscopic analysis of the R-loops formed between various DNA fragments and yeast mRNA, we could locate the ribosomal protein genes on the physical maps of the cloned DNA fragments. The R-loop structures observed indicate that a number of the ribosomal protein genes contain an intervening sequence.
Assuntos
Proteínas Ribossômicas/genética , Saccharomyces/genética , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , Genes , Hibridização de Ácido NucleicoRESUMO
A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.
Assuntos
DNA Fúngico/isolamento & purificação , Genes , Proteínas Ribossômicas/genética , Saccharomyces/genética , DNA Recombinante/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Hibridização de Ácido Nucleico , RNA Bacteriano/metabolismoRESUMO
In order to identify and to study the ribosomal protein genes in yeast we have tried to purify the mRNA coding for ribosomal proteins. Poly(A)-containing RNA from the yeast Saccharomyces carlsbergensis was fractionated according to size using preparative sucrose gradient centrifugation. The various (size) fractions were translated in vitro in a wheat germ cell-free system. The products were analysed by sodium dodecylsulfate/polyacrylamide gradient gel electrophoresis as well as by acetic acid/urea gel electrophoresis. It was found that an mRNA fraction of about 9 S directs the synthesis in vitro of proteins that have properties characteristic of ribosomal proteins, i.e. they are both small and basic. The ribosomal nature of these proteins was further established by two-dimensional gel electrophoresis. This small-size mRNA fraction can be used as a probe for the identification of ribosomal protein genes in recombinant DNA molecules.
Assuntos
Poli A/metabolismo , RNA Mensageiro/isolamento & purificação , Proteínas Ribossômicas/biossíntese , Saccharomyces cerevisiae/genética , Eletroforese em Gel de Poliacrilamida , RNA Mensageiro/genéticaRESUMO
The effects of ultraviolet irradiation on the rates of synthesis of individual ribosomal proteins in yeast were examined and compared with the ultraviolet sensitivities of the synthesis of other yeast proteins. It was found that the synthesis of yeast ribosomal proteins is much more sensitive to ultraviolet irradiation than that of other yeast cellular proteins. Taking into account the half-life of yeast mRNA, the results obtained indicate that the genes coding for ribosomal proteins form part of long transcriptional units, which are much longer than the DNA stretch needed to code for a ribosomal protein of average molecular weight. Saturation hybridization of total poly(A)-containing mRNA with yeast nuclear DNA revealed that as much as 30% of DNA is complementary to yeast mRNA. Thus, the primary transcript of a protein gene on the average is about 1.7 times the length of the actual messenger. On the basis of the various experimental data we suggest a clustering of the yeast ribosomal protein genes in a number of common transcriptional units.
