Polyadenylic acid on poliovirus RNA. II. poly(A) on intracellular RNAs.

The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.     

Ribosomal RNA genes of Saccharomyces cerevisiae. II. Physical map and nucleotide sequence of the 5 S ribosomal RNA gene and adjacent intergenic regions.

A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribosomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribosomal RNA. The nucleotide sequence of the DNA fragments carrying the 5 S ribosomal RNA gene and adjacent regions has been determined. The sequence unambiguously identifies the 5 S ribosomal RNA gene, determines its polarity within the ribosomal DNA repeating unit, and reveals the structure of its promoter and termination regions. Partial DNA sequence of the regions near the beginning and end of the 35 S ribosomal RNA gene has also been determined as a preliminary step in establishing the structure of promoter and termination regions for the 35 S ribosomal RNA gene.     

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.     

A method for the isolation of segments from the 5′ ends of retrovirus RNA

A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA.     

tRNA''s associated with the 70S RNA of avian myeloblastosis virus.

The distribtuion of various amino acid tRNA's in the 4S RNA components of avian myeloblastosis virus (AMV) and in 4S RNA prepared from chicken cmbryo cells, chicken myeloblasts, and chicken livers was determined. This was done by aminoacylating the 4S RNA samples with a mixture of 17 radioactive amino acids and subsequently identifying the tRNA-accepted amino acids on an amino acid analyzer after deacylation. In embryo cells, myeloblasts, and liver, tRNA's accepting all 1m amino acids were demonstrated. "Free" AMV 4S RNA was characterized by very low quantities of glutamate, valine, and tyrosine tRNA's. RNAs accepting all 17 amino acids, with the exception of tyrosine, were shown to be present in the "70S-associated" 4S RNA which dissociates at 60 C. The bulk of the 70S-associated 4S RNA was dissociated at 60 C at low ionic strength with a concomitant conversion of 70S RNA to 35S RNA. The residual associated 4S RNA was dissociated by further heating of the 35S RNA to 80 C; tryptophan tRNA accounted for greater than 90% of the total amino acid accepting activity in this fraction. The results support other studies in suggesting that tryptophan tRNA may serve as a primer for DNA synthesis in AMV, as has been shown in Rous sarcoma virus.     

Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs.

The organization of the ribosomal DNA repeating unit from Saccharomyces cerevisiae has been analyzed. A cloned ribosomal DNA repeating unit has been mapped with the restriction enzymes Xma 1, Kpn 1, HindIII, Xba 1, Bgl I + II, and EcoRI. The locations of the sequences which code for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs have been determined by hybridization of the purified RNA species with restriction endonuclease generated fragments of the repeating unit. The position of the 5.8 S ribosomal DNA sequences within the repeat was also established by sequencing the DNA which codes for 83 nucleotides at the 5' end of 5.8 S ribosomal RNA. The polarity of the 35 S ribosomal RNA precursor has been established by a combination of hybridization analysis and DNA sequence determination and is 5'-18 S, 5.8 S, 25 S-3'.     

Biochemical characterization of endogenous type C virus information in differentiated and undifferentiated murine teratocarcinoma-derived cell lines.

Undifferentiated teratocarcinoma cells express sixfold-higher levels of endogenous xenotropic type C virus-related RNA than differentiated cells. Three species of polyadenylated viral RNA (35S, 24S, and 14S) have been identified in the undifferentiated teratocarcinoma cells. Paradoxically, neither viral particles nor viral proteins have been detected in these cells.     

Purification and translation of murine mammary tumor virus mRNA's

We have studied the functions of the intracellular RNAs of mouse mammary tumor virus (MMTV) by purification and translation in vitro. Two major size classes of MMTV RNA, 35S and 24S RNA, were isolated from MMTV-infected rat (XC) cells and cultured mammary tumor cells by preparative hybridization of whole cell or polyadenylated RNA to cloned MMTV DNA covalently bound to chemically activated paper disks (diazobenzyloxymethyl paper). Genomic-length (35S) RNA was prepared free of 24S RNA by rate zonal sedimentation in sucrose gradients. Experiments using [3H]uridine-labeled cellular RNA indicated that the preparative annealing method was highly specific and capable of effecting a 300-fold enrichment for viral RNA; the recovered RNA appeared to be intact under denaturing conditions and directed synthesis of full-length gag and env polypeptides in vitro. The products of in vitro translation were identified by gel mobility, immunoprecipitation tests with antisera against gag and env products, and partial digestion with Staphylococcus V8 protease. The 35S RNA species directed synthesis of several gag-related polypeptides, including three previously reported in extracts of infected cells; 24S RNA directed synthesis of two polypeptides closely related to env proteins from infected cells. Therefore, 35S RNA includes mRNA's for gag and gag-pol, whereas 24S RNA is the mRNA for env. These results help establish the position of env on the physical map of the MMTV genome and bear upon the coding potential of the genome.     

NEWLY SYNTHESIZED RNA IN NUCLEI ISOLATED FROM NERVE AND GLIAL CELLS

—Nuclear RNA from neurones, astrocytes and other glial cells was pulse-labelled in vivo with [³H]uridine and analysed by sucrose density-gradient centrifugation after various periods of incorporation. Thirty min after the injection of the isotope, rapidly-labelled RNA appeared in all three cell types, a heterogeneous fraction sedimenting above 30S, the others at 25 and 12S. The transformation rate of the two latter components was equally rapid in all three types of nuclei studied. These components are assumed to be of messenger nature. The heavy fractions underwent transformations which in other cells have been described to lead to rRNA formation. The temporal pattern as well as the sequence of changes were similar in nuclei from neurones and astrocytes, the only difference being that a 35S intermediate was found in the former and a 32S in the latter. In non-astrocytic glial nuclei, synthesis and transformation of the 45S component were delayed as compared to the other cell types and the processing of this component may involve both a 32S and a 35S intermediate. Moreover, the radioactivity incorporated in all the nuclear RNA species was always lower in these cells.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre‐messenger RNAs in kinetoplastid protozoa are substrates of uridylate‐specific RNA editing. RNA editing converts non‐functional pre‐mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of ～20S and ～35–40S and present the three‐dimensional structures of both complexes by electron microscopy. The ～35–40S complexes consist of a platform density packed against a semispherical element. The ～20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The ～20S editosomes contain an RNA‐binding site, which binds gRNA, pre‐mRNA and gRNA/pre‐mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.     

Methionine-Dependent Synthesis of Ribosomal Ribonucleic Acid During Sporulation and Vegetative Growth of Saccharomyces cerevisiae

Methionine limitation during growth and sporulation of a methionine-requiring diploid of Saccharomyces cerevisiae causes two significant changes in the normal synthesis of ribonucleic acid (RNA). First, whereas 18S ribosomal RNA is produced, there is no significant accumulation of either 26S ribosomal RNA or 5.8S RNA. The effect of methionine on the accumulation of these RNA species occurs after the formation of a common 35S precursor molecule which is still observed in the absence of methionine. During sporulation, diploid strains of S. cerevisiae produce a stable, virtually unmethylated 20S RNA which has previously been shown to be largely homologous to methylated 18S ribosomal RNA. The appearance of this species is not affected by the presence or absence of methionine from sporulation medium. However, when exponentially growing vegetative cells are starved for methionine, unmethylated 20S RNA is found. The 20S RNA, which had previously been observed only in cells undergoing sporulation, accumulates at the same time as a methylated 18S RNA. These effects on ribosomal RNA synthesis are specific for methionine limitation, and are not observed if protein synthesis is inhibited by cycloheximide or if cells are starved for a carbon source or for another amino acid. The phenomena are not marker specific as analogous results have been obtained for both a methionine-requiring diploid homozygous for met13 and a diploid homozygous for met2. The results demonstrate that methylation of ribosomal RNA or other methionine-dependent events plays a critical role in the recognition and processing of ribosomal precursor RNA to the final mature species.