Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.

[1]  M. Case,et al.  Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa. , 1975, The Journal of biological chemistry.

[2]  M. Case,et al.  Genetic evidence on the organization and action of the qa-1 gene product: a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Case,et al.  Characterization of qa-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques , 1977, Journal of bacteriology.

[4]  U. Loening.  The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. , 1967, The Biochemical journal.

[5]  B. Paterson,et al.  Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Means,et al.  Use of Sepharose 4B for preparative scale fractionation of eukaryotic messenger RNA's. , 1974, Preparative biochemistry.

[7]  R. Schimke,et al.  Ovalbumin messenger RNA: evidence that the initial product of transcription is the same size as polysomal ovalbumin messenger. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Vanderhoef,et al.  Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa , 1975, Journal of bacteriology.

[9]  P. Mirkes,et al.  Synthesis of polyadenylic acid-containing ribonucleic acid during the germination of Neurospora crassa conidia , 1976, Journal of bacteriology.

[10]  J. Taylor,et al.  Synthesis of a deoxyribonucleic acid sequence complementary to ovalbumin messenger ribonucleic acid and quantification of ovalbumin genes. , 1973, The Journal of biological chemistry.

[11]  R. F. Manning,et al.  Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori. , 1976, Journal of molecular biology.

[12]  B. Groner,et al.  Length heterogeneity in the poly (adenylic acid) region of yeast messenger ribonucleic acid. , 1974, Biochemistry.

[13]  R. S. Chaleff.  The inducible quinate-shikimate catabolic pathway in Neurospora crassa: genetic organization. , 1974, Journal of general microbiology.

[14]  J. Bantle,et al.  Specificity of oligo (dT)-cellulose chromatography in the isolation of polyadenylated RNA. , 1976, Analytical biochemistry.

[15]  L. Kedes.  Histone messengers and histone genes , 1976, Cell.

[16]  M. Loo.  Neurospora crassa temperature-sensitive mutant apparently defective in protein synthesis , 1975, Journal of bacteriology.

[17]  R. Arceci,et al.  The programmed switch in lysine-rich histone synthesis at gastrulation , 1976, Cell.

[18]  H. Lodish,et al.  Different lifetimes of reticulocyte messenger RNA , 1976, Cell.

[19]  H. Soreq,et al.  Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes. , 1976, European journal of biochemistry.

[20]  J. Piatigorsky,et al.  Quantitation of delta-crystallin messenger RNA during lens induction in chick embryos. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[21]  George A. Bray,et al.  A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter , 1960 .

[22]  W. Gratzer,et al.  Electrophoresis of RNA in formamide. , 1974, Biochemistry.

[23]  B. Groner,et al.  Polyadenylate metabolism in the nuclei and cytoplasm of Saccharomyces cerevisiae. , 1975, The Journal of biological chemistry.

[24]  H. J. Baker,et al.  In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA. , 1976, The Journal of biological chemistry.

[25]  C Baglioni,et al.  Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Warner,et al.  Polyadenylic acid sequences in yeast messenger ribonucleic acid. , 1973, The Journal of biological chemistry.

[27]  D. Wallach,et al.  Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. , 1971, Biochemistry.

[28]  M. Case,et al.  Genetic regulation of Quinate-Shikimate Catabolism in Neurospora Crassa , 1973 .

[29]  M. Edelman,et al.  Poly(A)-associated RNA from the mitochondrial fraction of the fungus Trichoderma. , 1976, European journal of biochemistry.

[30]  R. Schwartz,et al.  Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin. , 1976, The Journal of biological chemistry.

[31]  M. Case,et al.  Constitutive mutants in a regulatory gene exerting positive control of quinic acid catabolism in Neurospora crassa. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Case,et al.  Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase , 1975, Journal of bacteriology.