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.     

Effects of RNase and RNA on in vitro aster assembly

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polucytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing condition. These results demonstrate that specfic polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.     

Nucleotide sequence of 5S ribosomal RNA from rainbow trout (Salmo gairdnerii) liver.

Staring from low molecular weight RNA obtained from rainbow trout (Salmo gairdnerii) liver, 5S ribosomal RNA (rRNA) was highly purified by successive chromatography on columns of DEAE-Sephadex A50 and Sephadex G100. Products of complete and partial digestions on this RNA with pancreatic ribonuclease (RNase A) [EC 3.1.4.22] and RNase T [EC 3.1.4.8] were isolated and sequenced by conventional and high-performance liquid chromatography (HPLC) procedures. The nucleotide sequence of this RNA thus established was compared with those of five other vertebrae 5S rRNAs, and the rates of base substitution per site per year were found to be nearly constant in these RNAs. The analyses of the partial digests of the trout 5S rRNA revealed several sites susceptible to RNase attack, which could be accounted for by the secondary structure model for eukaryotic 5S rRNAs proposed by Nishikawa and Takemura (1).     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

Satellite RNA of cucumber mosaic virus forms a secondary structure with partial 3''-terminal homology to genomal RNAs.

Sat-RNA is one of several replicating satellite RNAs which have been isolated from RNA encapsidated in cucumber mosaic virus (CMV) and which are totally dependent on CMV for replication. The 336 residue sequence of Sat-RNA obtained using the dideoxynucleotide chain termination and partial enzymic digestion procedures shows only a few short stretches (up to 11 residues) of sequence homology with one of the three CMV genomal RNAs so far sequenced. Sat-RNA has 88% sequence homology with another, previously sequenced, satellite RNA of CMV, CARNA 5. Analysis of partial digests of 5'- or 3' -32P-Sat-RNA with nuclease S1 or RNase T1 under non-denaturing conditions showed that only about 10% of the residues in Sat-RNA were cleaved. Further data on base-paired segments of Sat-RNA were obtained using digestion with RNase T1 followed by electrophoretic fractionation of the resulting fragments under both non-denaturing and denaturing conditions. On the basis of this data, a complete secondary structure model is proposed for Sat-RNA with 52% of its residues involved in base pairs. A prominent hairpin at the 3'-terminus of Sat-RNA shows considerable sequence and structural homology with parts of the 3'-terminal tRNA-like structure of the CMV genomal RNAs.     

Oligonucleotide mapping of non-radioactive virus and messenger RNAs.

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.     

RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNA''s.

The procaryotic RNA processing enzyme RNase III (endoribonuclease III [EC 3.1.4.24]) was used to probe vesicular stomatitis virus (VSV) RNAs for specific sites that could be recognized and cleaved. The effect of the enzyme on the RNAs was monitored by measuring their subsequent migration in denaturing agarose-urea gels. VSV virion RNA (negative strand; Mr, 4 X 10(6)) was cleaved by the enzyme to yield a set of discrete fragments which ranged on size from 3.5 X 10(6) to 0.2 X 10(6) daltons. The cleavage was a function of enzyme concentration, salt concentration, and time. A maximum of 20 to 22 fragments was generated under conditions of low enzyme concentration or short times of incubation. VSV genome-length intracellular RNA of both + and - polarity was also cleaved by RNase III. In contrast to the findings with virion-length RNA, however, the migration rates of VSV mRNA's purified by chromatography on polyuridylic acid-Sepharose were unaffected by treatment with RNase III. These results show that specific sites in the virion RNA and its full-length complement can be recognized by RNase III. Sites of this type are not present in the polyadenylic acid-containing mRNA, however.     

Recent developments in methods for RNA sequencing using in vitro 32P-labeling

A variety of approaches that utilize in vitro 32P-labeling of RNA and of oligonucleotides in the sequence analysis of RNAs are described. These include 1) methods for 5'- and 3'- end labeling of RNAs; 2) end labeling and sequencing of oligonucleotides present in complete T1 RNase or pancreatic RNase digests of RNA; 3) use of random endonucleases, such as nuclease P1, for terminal sequence analysis of end labeled RNAs; and 4) use of base specific enzymes or chemical reagents in the sequence analysis of end-labeled RNAs. Also described is an approach to RNA sequencing, applied so far to tRNAs, which is based on partial and random alkaline cleavage of an RNA to generate a series of overlapping oligonucleotide fragments, all containing the original 3'-end of the RNA. Analysis of the 5'- end group of each of these oligonucleotides (following 5'-end labeling with 32P) provides the sequence of most of the tRNA. The above methods have been used to derive the sequences of several tRNAs, the ribosomal 5S and 5 x 8S RNAs, a viroid RNA, and large segments of both prokaryotic and eukaryotic ribosomal and messenger RNAs.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

In-vitro translation of messenger-RNA from developing bean leaves. Evidence for the existence of stored messenger-RNA and its light-induced mobilisation into polyribosomes

Poly(A)-containing messenger RNA was purified from polyribosomes isolated from the primary leaves of 7-day-old dark-grown seedlings of Phaseolus vulgaris var. Masterpiece. Analysis of the messenger RNA on 2.4% polyacrylamide gels showed that it consists of a heterogeneous population of molecules with an average molecular weight of 500,000. The nucleotide composition of the RNA was 16.0% cytidylic acid, 39.4% adenylic acid, 21.3% guanylic acid and 23.2% uridylic acid. Based on the degree of resistance of the RNA to digestion with ribonucleases A and T₁ the average length of the poly(A) sequence was calculated to be 120 nucleotides. No significant differences in mobility in polyacrylamide gels, nucleotide composition or polyadenylic acid content were found between the poly(A)-containing mRNA from polyribosomes of primary leaves of dark-grown plants and those given a 16 h white light treatment. Purified poly(A)-containing mRNA was shown to direct the incorporation of [³⁵S]methionine into proteins in an in vitro protein-synthesising system from wheat germ. The protein products were fractionated according to molecular size by electrophoresis in 15% polyacrylamide/urea/SDS gels and the protein bands were detected by fluorography. Messenger RNAs directing the synthesis of three polypeptides with molecular weights of 34,000, 32,000 and 25,000 were detected in polyribosomes of plants following white light treatment. These messenger RNAs were absent, or present in much lower amounts, in polyribosomal messenger RNA from leaves of dark-grown plants, although they were present in total cell poly(A)-containing RNA. This indicates that certain messenger RNAs may be stored in the dark and that light stimulates these RNAs to engage in polyribosome formation. Continuous far-red (730 nm) irradiation for 4 h also caused the appearance of these messenger RNAs in the polyribosomes although 5 min red light followed by 4 h darkness had little effect. This suggests that phytochrome acting in the high energy mode, may be the photoreceptor responsible for initiating the response.Abbreviations mRNA messenger-RNA - rRNA ribosomal RNA - oligo (dT) oligo (deoxythymidylic acid) - poly(A) polyadenylic acid - EDTA ethylenediamine-tetra-acetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - SDS sodium dodecyl sulphate     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.     

Polyadenylic acid sequences in the RNA of Hyphomicrobium

Heterogeneous RNA containing polyadenylic acid [poly(A)] sequence has been isolated from Hyphomicrobium by affinity chromatography on oligothymidylic acid cellulose and polyuridylic acid Sepharose columns. About 0.1 to 0.3% of [3H]adenine-labeled RNA over a 60-min period is associated with poly(A) sequences. This percentage decreases to about 0.03 in a 20-h labeling period. The poly(A) tracts recovered after digestion with ribonuclease A and T1 are composed of greater than 95% adenine residues and are up to 200 nucleotides in length with a predominant range of 15 to 40 nucleotides. Adenosine and AMP are present in the ratio of 1:36 in alkaline digests of Hyphomicrobium poly(A) tracts. This is compatible with nucleotide lengths determined on acrylamide gels and location at the 3'-OH terminus of the RNA molecule.     

Size and location of poly (A) in encephalomyocarditis virus RNA.

Encephalomyocarditis (EMC) virus RNA contains a covalently bound sequence of polyriboadenylic acid (poly(A). This was determined by two-dimensional gel electrophoresis of complete T1 and pancreatic RNase digests of formamidesucrose gradient-purified RNA and subsequent analysis of the product by alkaline hydrolysis. The size of the EMC virus genomic poly(A) sequence was estimated by formamide-polyacrylamide gel electrophoresis of the RNase-resistant product, or by [3H-]poly(U) hybridization to freshly purified virion RNA, to be, on average, 40 nucleotides in length. The evidence obtained from [3H-]isoniazid labelling and other experiments would indicate that the poly(A) sequence is located at the 3'-terminus of EMC virus RNA.     

Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA.

The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large T1-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and T1; (iii) eight more which were isolated from hybrids treated with RNases A and T1; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLV's including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid coordinate of SFFV RNA were deduced from the size of the smallest polyadenylic acid-tagged RNA fragment from which a given oligonucleotide was isolated. The resulting oligonucleotide map could be divided roughly into three segments: two terminal segments which are mosaics of oligonucleotides of classes i, ii, and iii and an internal segment between 2 and 2.5 kilobases from the 3' end containing the two oligonucleotides shared with amphotropic MLVs. Since SFFV RNA consists predominantly of sequence elements related to ecotropic and amphotropic helper-independent MLVs, it would appear that the transforming gene of SFFV is not a major specific sequence unrelated to genes of helper viruses, as is the case with Rous sarcoma and probably withe other defective sarcoma and acute leukemia viruses.