Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.     

Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells.

A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.     

Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus.

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.     

Adenovirus type 2 late mRNA's: structural evidence for 3'-coterminal species.

Adenovirus type 2-infected HeLa cells were labeled with 32PO4 during the period 14 to 17 h postinfection. Viral mRNA's with polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fractionated according to size by electrophoresis through an acrylamide-agarose slab gel. Messenger bands were eluted and partially degraded with alkali. RNA fragments from each band that contain polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fingerprinted two-dimensionally after T1 RNase digestion. Three bands, with mobilities of approximately 26S, 21S, and 18S, shared two large characteristic T1 oligonucleotides in common in the fingerprints of their 3'-terminal sequences. These oligonucleotides were mapped with a Hpa II restriction fragment of adenovirus type 2 DNA with coordinates 49-50.2. We conclude that the three mRNA's are coterminal in sequence at their 3' ends and overlap at internal positions. Implications for the protein-coding potential of these mRNA's and the mechanisms of adenovirus tyep 2 late RNA processing are discussed.     

Properties of a human liver ribonuclease. Inhibition by polynucleotides and specificity for phosphodiester bond cleavage to yield purine nucleosides at the 5' termini.

A ribonuclease, purified 2500-fold from human liver, was found to be inactive against synthetic homopolynucleotides, whereas synthetic co-polymers containing adenylic acid were rapidly degraded. The specificity of the RNase is unique in that only purine residues, in a 5:4 ratio of guanylic to adenylic acid, are found at the 5' termini of the degradation products of yeast RNA. No specificity was observed at the 3' termini of the fragments. When analyzed by DEAE-cellulose chromatography, approximately 80% of the oligonucletoides were 4 to 11 residues in length. The hydrolysis of RNA by the liver enzyme, when examined in low ionic strength buffer, could be increased severalfold over control levels by the addition of polyamines. The enzyme was found to exist as two distinct species on sucrose gradients, with molecular weights of 128,000 and 14,000. However, the addition of spermidine to the gradients resulted in the recovery of all the enzyme activity as the smaller species. The polyamines were also shown to reverse the inhibition of the enzyme by the ordered polynucleotides, polyguanylic acid and polyadenylic acid. Inhibition of enzyme activity by the polyadenylic acid segment of various mammalian mRNAs was also demonstrated.     

Gene mapping by fluorescent in situ hybridization

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.     

Polyadenylic acid in the genomic RNA of mengovirus.

The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosine-labeled 35S viral RNA.     

Replicase gene of coxsackievirus B3.

A cDNA copy covering two-thirds of the coxsackievirus B3 genome was cloned in the PstI site of the pBR322 vector. A nucleotide sequence containing the gene for the viral replicase and the 3' noncoding region of the coxsackievirus B3 genome was determined. The predicted amino acid sequence of the coxsackievirus B3 replicase was shown to be remarkably similar to that of the poliovirus 1 replicase. The 3' noncoding region, in contrast, was only weakly homologous to the poliovirus 1 sequence but showed a close relationship to the sequence of swine vesicular disease virus, a variant of coxsackievirus B5. A 13-nucleotide-long segment located near the polyadenylic acid junction is conserved in several members of the enterovirus group and may thus serve an important function during replication of viral RNA.     

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.     

Characterization of the 3' Termini of the RNAs of Cowpea Mosaic Virus

A sequence of polyadenylic acid, homogeneous in composition but heterogeneous in length, was isolated from complete pancreatic RNase digests of both middle and bottom RNAs of cowpea mosaic virus. The polyadenylic acid was 3′-terminal and occurred once per molecule. A fragment consisting of the polyadenylic acid and approximately the next 25 nucleotides could be isolated from complete T1 RNase digests of either RNA. The region adjacent to the polyadenylic acid in both RNAs was rich in pyrimidines. The mobilities of the fragments in 12.4% polyacrylamide-8 M urea gels were used to estimate their lengths and to calculate number average and weight average molecular weights.     

Biochemical properties and hormonal regulation of barley nuclease

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.     

Synthesis of polyadenylic acid-containing ribonucleic acid during the germination of Neurospora crassa conidia.

Ribonucleic acid (RNA) synthesized during the first 1 h of conidial germination (15 to 20, 25 to 30, and 55 to 60 min) has been characterized by sucrose-sodium dodecyl sulfate gradient centrifugation, binding to polyuridylic acid filters, and oligo(dT)-cellulose chromatography. At all labeling periods examined, polyadenylic acid-containing RNA is synthesized, processed, and incorporated into polysomes. Approximately 40% of the labeled RNA sedimenting between 5 and 17S binds to polyuridylic acid filters. RNA which binds to oligo(dT)-cellulose displays a heterogeneous distribution in sucrose-sodium dodecyl sulfate gradients with a major, broad peak at 10-16S. In addition, some polyadenylic acid-containing RNA sediments beyond the 25S marker. Approximately 3% of the [3H]adenosine in pulse-labeled polysomal RNA is in polyadenylic acid segments resistant to pancreatic and T1 ribonucleases.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA''s of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.