Multiple Genetic Components in Bromegrass Mosaic Virus

Bromegrass mosaic virus has three types of structurally similar virions all of which are required for infectivity. The virions contain either one RNA molecule of molecular weight 1.09×10⁶ or 0.99×10⁶ or two of molecular weights 0.75×10⁶ and 0.28×10⁶. A mixture of the isolated RNAs is infectious and deletion of any but the smallest greatly reduces infectivity. The third largest RNA component contains the coat protein gene.     

Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region.

The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 10⁶. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T₁-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Multiple forms of human dihydrofolate reductase messenger RNA: Cloning and Expression in Escherichia coli of their DNA coding sequence

The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA₂-B). A major fraction of this promoting activity is associated with a 3.8 × 10³ base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA₂-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 10³ bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 10³ base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 10³, 1.0 × 10³ and 0.8 × 10³ bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA₂B and HeLa BU25.     

Purification and Properties of the Geminivirus Euphorbia Mosaic Virus

Euphorbia mosaic virus was purified from infected plants of Nicotiana benthamiana. Highest concentrations of virus particles were found in infected plant tissue between 10–12 days after inoculation. The enzyme driselase assisted in purification of the virus particles from the infected tissue yielding about 600 μg/kg of plant material. Purified preparations showed a maximum absorption at 260–263 nm and the ratio of absorption at 260 and 280 nm was 1.4. The viral nucleic acid was digestedby DNase I and S₁ Nuclease but not RNase A. A single coat protein with a MW of 32,000 d and two DNA bands with a MW 0.96 × 10⁶ d (2870 nucleotides) and 0.90 × 10⁶ d (2700 nucleotides) were associated with the purified virus particles. Virus specific DNA was isolated from infected tissue between 7 and 15 days after inoculations.     

Properties of a tomato isolate of Pelargonium zonate spot virus

A manually transmissible virus isolated from tomato plants with stunting, unfruitfulness, malformation and yellow rings and line patterns of the leaves was indistinguishable from Pelargonium zonate spot virus (PZSV) in biological, physico-chemical and serological properties. The tomato isolate (PZSV-T) of PZSV was seed transmitted in Nicotiana glutinosa and was detected in the pollen of this host. In sap of N. glutinosa PZSV-T lost infectivity after diluting 10^-1 to 10^-2, heating for 10 min at 35 to 40 °C or storage at 25 °C for 7 h. Virus particles were quasi-spherical with a diameter ranging between 25 and 35 nm with a modal value of 29 nm. Particles sedimented as three components (TV, MV and BV) with sedimentation coefficients of 80S (TV), 90S (MV) and 118S (BV); component BV is probably an aggregate of TV. Particles were unstable in CsCl and CS₂SO₄ but formaldehyde-stabilised particles banded at a common density of 1–268 g/cm³ in Cs₂SO₄. Particles contained a single protein species with mol. wt of c. 23000 and c. 18% single stranded RNA present as two species with mol. wts of c. 1.25 × 10⁶ (RNA-1) and 0.95 × 10⁶ (RNA-2). Mixtures of RNA-1 + RNA-2 were infectious and this infectivity was not enhanced by the addition of coat protein. Virus particles had a Tf (mid point of extinction when heated) of 63 °C and were readily dissociated by 0.1% SDS. PZSV-T was serologically unrelated to alfalfa mosaic and to 32 isometric viruses including five ilarviruses. Some properties of PZSV resemble those of ilarviruses but others are sufficiently different to suggest that it may not be a member of this virus group.     

Structural features unique to the 5 S ribosomal RNAs of the thermophilic cyanobacterium Synechococcus lividus III and the green plant chloroplasts

The complete nucleotide sequence of the 5 S ribosomal RNA from the thermophilic cyanobacterium Synechococcus lividus III was determined. The sequence is: ^5′U-C- C-U-G-G-U-G-G-U-G-A-U-G-G-C-G-A-U-G-U-G-G-A-C-C-C-A-C-A-C-U-C-A-U-C- C-A-U-C-C-C-G-A-A-C-U-G-A-G-U-G-G-U-G-A-A-A-C-G-C-A-U-U-U-G-C-G-G-C- G-A-C-G-A-U-A-G-U-U-G-G-A-G-G-G-U-A-G-C-C-U-C-C-U-G-U-C-A-A-A-A-U-A- G-C-U-A-A-C-C-G-C-C-A-G-G-G-U_OH^3′This 5 S RNA has regional structural characteristics that are found in the green plant chloroplast 5 S RNAs and not in other known sequences of 5 S ribosomal RNAs. These homologies suggest a close phylogenetic relationship between S. lividus and the green plant chloroplasts.     

Some properties of an isolate of tobacco rattle virus from Northern Ireland

A virus obtained from soil in which potato plants had shown severe spraing symptoms induced symptoms on indicator plants typical of tobacco rattle virus (TRY). Purified virus preparations of a local-lesion isolate contained particles of two modal lengths, 192 nm and 94 nm containing RNA molecules of mol. wt 2.4 × 10⁶ and 1.23 × 10⁶. Virus coat protein had a mol. wt of c. 21 500. The virus was serologically distantly related to TRY (SYM) and pea early browning virus (PEBV) SP5, but did not react with TRY (CAM) or TRY (PRN) antisera. However, cDNA hybridisation indicated that the virus was more closely related to TRY (PRN) than either TRY (SYM) or PEBV (SP5). The virus isolate has been designated TRY (NI).     

Characterization of a Latent Elongated Virus from Globe Artichoke (Cynara scolymus L.) in Italy

A virus with filamentous particles was isolated from symptomless plants of Cynara scolymus cvs Romanesco and Terom obtained by in vitro meristem culture in northern Italy. The virus was characterized biologically, physico-chemically and serologically. The cytopathology induced by its infection in two artificial hosts (Chenopodium quinoa and Nicotiana benthamiana) was also investigated. The virus has slightly flexuous elongated particles measuring 12 ± 664 nm; its sedimentation coefficient, RNA content, mol. wts. of RNA and coat protein subunits are 150 S, 6 %, 2.2 × 10⁶ and 2.9 × 10⁴, respectively. In microprecipitation tests, it resulted serologically related to poplar mosaic virus (PopMV) (SDI = 4–5). Cellular inclusions and cytopathology observed in both the artificial hosts conform to those of the carlavirus group.     

Characterization of Virion RNAs of Southern Bean Mosaic Virus by Electron Microscopy

Electron microscopy of denatured RNA of southern bean mosaic virus (SBMV) shows two principal linear components, 0.31 ± 0.08 μm (subgenomic RNAs, MW 0.51 × 10⁶) and 0.80 ± 0.17 μm (genomic RNA, MW 1.3 × 10⁶ 25S). Nondenatured RNA (～ 32S) from heat-inactivated virions measure 1.0 ± 0.20 μm (MW 1.64 × 10⁶) but is poorly-infectious. Upon denaturation the 325 RNA disaggregates into components of lengths typical of the genomic and subgenornic RNAs and infectivity is restored.     

Characterization of sub-genomic double-stranded RNAs from virus-infected plants

Double-stranded RNAs (sub-RFs) smaller than the double-stranded RNAs (RFs) corresponding to genomic RNAs of tobacco mosaic (TMV) and cowpea chlorotic mottle (CCMV) viruses were isolated from infected plants and characterized. Seven of the 12 sub-RFs of TMV that were found ranging in size from 3.00 – 0.42 × 10⁶ daltons corresponded to twice the size of the 7 sub-genomic mRNAs reported by Goelet and Karn (8). Six sub-RFs of CCMV were found ranging from 0.98 – 0.39 × 10⁶ daltons with the most abundant species corresponding to twice the size of RNA 4. The kinetics of incorporation of ³H-uridine into sub-RFs were different from that into RFs. Incorporation into sub-RFs was slow and linear whereas that into RF turned over rapidly.     

Plant 5.8S RNA is a Component of 80S but not 70S Ribosomes

LIVING organisms contain two classes of ribosomes that can be distinguished by differences in their size, the molecular weights of their constituent high molecular weight RNAs and their sensitivity to certain inhibitors of protein synthesis. The ribosomes of one type occur in bacteria, blue-green algae and chloroplasts. They have sedimentation coefficients of approximately 70S^1,2, contain RNA with molecular weights of about 1.1 × 10⁶ (23S) and 0.56 × 10⁶ (16S)³ and their activity is inhibited by chloramphenicol^4,5, lincomycin and spectinomycin⁶. The ribosomes of the other type are found in the cytoplasm of animal and plant cells, have sedimentation coefficients of 80S^1,2, contain RNA with molecular weights of 1.3?1.75 × 10⁶ (25–28S) and 0.7 × 10⁶ (18S)³ and are prevented from functioning by cycloheximide⁴.     

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses.

The genome of avian sarcoma virus (ASV) contains four known genes: gag, encoding structural proteins of the viral core; pol, encoding the viral RNA-directed DNA polymerase; env, encoding the glycoprotein(s) of the viral envelope; and src, which is responsible for neoplastic transformation of the host cell. We have located these genes on virus-specific RNAs in cells productively infected with both nondefective and defective strains of ASV by using molecular hybridization with DNAs complementary to specific portions of the ASV genome.The cytoplasm of cells producing nondefective ASV contains three species of polyadenylated virus-specific RNA, each of which has chemical polarity identical to that of the viral genome. The largest species has a molecular weight of 3.3 × 10⁶ daltons and a sedimentation coefficient of 38S, encodes all four viral genes, and is probably identical to the viral genome. A second species has a molecular weight of 1.8 × 10⁶ daltons and a sedimentation coefficient of 28S, and encodes the 3′ half of the viral genome, including env, src and a genetically silent region known as “c.” The smallest species has a molecular weight of 1.2 × 10⁶ daltons and a sedimentation coefficient of 21S, and encodes only src and “c.” All three species of virus-specific RNA contain nucleotide sequences at least partially homologous to a sequence of 101 nucleotides found at the extreme 5′ end of the ASV genome. This sequence may not be present in the portions of the ASV genome which encode the 28S and 21S virus-specific RNAs, and hence may be joined to these RNAs during their maturation from precursor molecules.The size and genetic composition of virus-specific RNAs in cells producing defective deletion mutants reflect the nature of the deletion. Deletions of either src or env eliminate the 28S virus-specific RNA, leaving a 21S RNA (which contains either env and “c” in the case of src deletions or src and “c” in the case of env deletions) and a 35S RNA which is probably identical to the viral genome.Based on these and related results, we propose a model for viral gene expression which conforms to previous suggestions that eucaryotic cells initiate translations only at the 5′ termini of messenger RNAs.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

Recognition of template RNA by Q polymerase: sequence at the 3'-terminus of Q 6S RNA

The major 3′-terminal sequences of Qβ 6S RNA have been determined by a combination of 3′-terminal labeling with ³H via the periodate-borohydride procedure, labeling of specific bases using ¹⁴C-labeled triphosphates and by ribonuclease T₁ digestion. The predominant sequence was GpCpCpA_OH with lesser amounts of GpCpC_OH and GpCpCpG_OH. Since the sole 5′-terminal base of 6S RNA is G, these results provide another example of the ability of Qβ polymerase to add a noncomplementary adenosine to the 3′-end and the first example of an ability to add a guanosine. Thus, all major sequences found may be considered derivatives of the sequence GpCpC_OH. This sequence differs significantly from those of other Qβ polymerase templates studied thus far, and thus reaffirms the requirement for additional internal structural features by which Qβ polymerase recognizes its templates.