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PubMed2000

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus.

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

Base Sequence↗

PubMed1996

The 126- and 183-kilodalton proteins of tobacco mosaic virus, and not their common nucleotide sequence, control mosaic symptom formation in tobacco.

Nucleotide substitutions at two positions within the open reading frame encoding the 126-kDa protein in the attenuated masked (M) strain of tobacco mosaic tobamovirus (TMV) to those found in the virulent U1-TMV genome led to the induction of near U1-TMV-like symptoms on leaves of Nicotiana tabacum L. cv. Xanthi nn by progeny virus (M. H. Shintaku, S. A. Carter, Y. Bao, and R. S. Nelson, Virology 221:218-225, 1996). In this study, further site-directed mutations were made at these positions within the M strain cDNA to determine whether the protein or nucleotide sequence directly controlled the symptom phenotype. The protein and not the nucleotide sequence directly controlled the symptom phenotype when amino acid 360 within the 126-kDa protein sequence was altered and likely controlled the symptom phenotype when amino acid 601 was altered. The effects of the substitutions at amino acid position 360 on viral protein accumulation were studied by pulse-labeling proteins in infected protoplasts. Accumulation of the 126- and 183-kDa proteins was less for an attenuated mutant than for two virulent mutants, but the viral movement protein and coat protein accumulated to levels reported to be sufficient for normal systemic symptom development. The size of necrotic local lesions on N. tabacum L. cv. Xanthi NN was negatively correlated with symptom development and accumulation of the 126-kDa protein for these mutants. With reference to this last finding, an explanation of the cause of the differing symptoms induced by these viruses is presented.

Amino Acid Sequence↗

PubMed1996

An infant with a mosaic 45,X/46,X,psu dic(Y) (pter-->q11.2::q11.2-->pter) karyotype and mixed gonadal dysgenesis studied for extent of mosaicism in the gonads.

AN infant with mixed gonadal dysgenesis was found to have a 45,X/46,Xpsu dic(Y) karyotype. A low level (8%) of mosaicism for the dic(Y) cell line was observed in peripheral blood lymphocytes and skin fibroblasts. The dicentric nature of the Y chromosome became apparent in fluorescence in situ hybridization studies. The presence of Y centromeric sequences was demonstrated in the paraffin-embedded testis and streak ovary sections. The ration of Y-positive cells was higher in the testis than in the streak ovary.

Chromosome Aberrations↗

PubMed1988

Transgenic tobacco expressing tobacco streak virus or mutated alfalfa mosaic virus coat protein does not cross-protect against alfalfa mosaic virus infection.

Transgenic tobacco plants expressing the coat protein (CP) genes of tobacco streak virus (TSV) and alfalfa mosaic virus (AIMV) were used in studies on cross-protection and genome activation. Plants expressing the TSV CP gene were highly resistant to infection with TSV nucleoproteins but were susceptible to infection with AIMV nucleoproteins. Moreover, these plants could be infected with a mixture of AIMV RNAs 1, 2, and 3 in contrast to the nontransformed control plants. This demonstrates that the endogenously produced TSV CP is able to activate the AIMV genome but does not cross-protect against this virus. Conversely, it was shown that plants expressing the AIMV CP gene did not resist TSV infection. Transgenic tobacco plants transformed with an AIMV CP gene with a frame-shift mutation in the reading frame were found to accumulate viral transcripts to a level similar to that obtained in plants expressing a wild-type AIMV CP gene. However, these plants did not produce detectable amounts of viral protein and showed no resistance to infection with AIMV nucleoproteins in contrast to transgenic plants accumulating wild-type AIMV CP. This demonstrates that it is the CP that is responsible for cross-protection in transgenic plants and not the chimeric CP mRNA.

Capsid↗

PubMed1989

Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.

Molecular Weight↗

PubMed1956

Studies on the aggregation reactions and basic dye binding of tobacco mosaic virus. I. Variation of pH, particle asymmetry, acid and base titration results, irreversible binding of methylene blue, ultraviolet absorption, and extent of heat denaturation in tobacco mosaic virus solutions with time of standing.

1. Aqueous solutions of tobacco mosaic virus were found to undergo a number of spontaneous changes on standing in the cold. The results of pH measurements, acid and base titrations, intrinsic viscosity determinations, studies on the irreversible binding of methylene blue with the virus, ultraviolet absorption, and the extent of nucleic acid splitting by heat denaturation indicated the occurrence of two successive reactions, the first one causing the release of hydrogen ions and a greater lability of the nucleic acid, and the second one, which involved end-to-end dimerization and which took place after 8 days of standing, requiring hydrogen ions. 2. The first over-all reaction was found to be a mixture of various types of reversible disaggregation and aggregation reactions, the nature of which depended on the pretreatment, the TMV concentration, the time of standing, and the phosphate concentration. For longer times of standing at high protein concentration a sudden drop in ultraviolet absorption is noted after dilution; also the drops in viscosity and pH are largest with a steep rise following, indicating the greatest breakup of end-to-end aggregates with formation of the side-to-side type. For concentrated solutions of TMV in water which have not stood long no drop in ultraviolet absorption is noted on dilution; the decrease in the other quantities is less, indicating that only a less extensive breakdown of end-to-end aggregates occurs. Addition of phosphate to concentrated solutions of TMV causes formation of side-to-side aggregates which break up on dilution. 3. Using the results for the pH increase and the viscosity increase in a given time interval for a given TMV preparation and also the slope of the corresponding titration curve at the pH mean, a value for the number of hydrogen ions taken up per TMV monomer in the formation of the end-to-end dimer was finally calculated. The average result obtained for two preparations was 3300. 4. Methylene blue, in the polymeric form, was demonstrated to cause complete irreversible conversion of TMV monomers to end-to-end dimers. At dye concentrations above 10(-4)M, higher TMV polymers are formed, but these are broken down to dimers on removal of free dye by dialysis. The irreversible binding ratios were shown to be decreased in accordance with the extent of the end-to-end aggregation of the preparation at the time of the experiment, which is in agreement with the concept that the irreversibly bound dye polymers go into the junction formed between two interacting TMV monomers. On the basis that only the monomers initially present in solution can react, maximum binding ratios corresponding to complete conversion of monomers to dimers were calculated from the observed irreversible binding ratios and from the fraction of dimers initially present which was obtained from viscosity data. The average result for three preparations in different states of aggregation was calculated to be 6565 for tetrameric binding or 3230 for dimeric binding, which agrees closely with the result obtained for the uptake of hydrogen ions per TMV monomer in the spontaneous dimerization.

Coloring Agents↗

PubMed1983

Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs.

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.

Brassica↗

PubMed1981

A boy with true hermaphroditism and sex chromosome mosaicism and a fertile woman with Turner mosaicism in a family with a translocation 8p:19P.

In a family with a balanced translocation t(8;19)(p21p13), there was a boy with true hermaphroditism and a karyotype 46,XX/46XY, t(8p;19p), and a woman with Turner mosaicism 46,XX, t(8p;19p). Both of them had whole body chimerism, which in the boy and possibly also in woman was due to the occurrence of double fertilization followed by fusion of the zygotes. The pathogenetic importance of the translocation for the development of these aberrations, and the clinical picture in the two patients are discussed.

Child, Preschool↗

PubMed1996

Trisomy 18 mosaicism associated with secondary amenorrhea: ratios of mosaicism in different samples and complications.

A 28-year-old woman who complained of irregular menstruation was diagnosed as suffering from trisomy 18 mosaicism. She was karyotyped because of her characteristic face, mild mental retardation and aberrant hyperpigmentation of the skin. Her motor function was within normal range. Physical and laboratory examinations, however, revealed obesity, short stature, minor anomalies of the fingers, many areas of hyperpigmentation on the trunk and the hips, hypergonadotropinemia, diabetes mellitus, liver dysfunction, and hyperlipidemia. The ratios of normal/trisomy 18 were 4:135 in blood lymphocytes, 3:11 in a hyperpigmented area of the skin, 20:0 in a normally pigmented area of the skin, and 14:6 in ascitic cells. Laparoscopy revealed that her ovaries contained neither follicles nor germ cells.

Abnormalities, Multiple↗

PubMed1983

Evidence for the presence of a hole in the capsid of turnip yellow mosaic virus after RNA release by freezing and thawing. Decapsidation of turnip yellow mosaic virus in vitro.

Turnip yellow mosaic virus (TYMV) RNA escapes from viral capsids after freezing and thawing the virus, and the remaining capsids look very similar to natural capsids in the electron microscope after negative staining [Katouzian-Safadi, M., Favre, A., and Haenni, A. L. (1980) Eur. J. Biochem. 112, 478-486]. In order to understand how an RNA of 2 X 10(6) Da (33% virus by weight) can escape from a compact protein shell we have compared artificial capsids formed after freezing TYMV and natural capsids produced in vivo in infected plants. We have used various physicochemical techniques including analytical ultracentrifugation, X-ray scattering, X-ray diffraction and orientation in a magnetic field. From the combination of these results we conclude that the escape of the RNA is accompanied by the formation of a hole in the capsid surface. The size of such a hole is estimated to 5-9 coat protein subunits.

Capsid↗

PubMed2001

Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae.

Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.

Alleles↗

PubMed1985

Mosaic and non-mosaic trisomy 15q2.

Two unrelated patients are presented. In the first mosaicism with normal cells and cells trisomic for the distal long arm (q2) of chromosome 15 was found. The 15q2 trisomy was due to a chromosome 14, to the long arm of which an extra 15q2 region was attached (14pter----14q32::15q22----15qter). In the second trisomy 15q2 was present as a consequence of a balanced t(7;15)(p22;q15) translocation in the mother.

Chromosome Banding↗

PubMedDate not supplied

[Directed fragmentation of tobacco mosaic virus RNA: excision of 3' terminal sections, containing coat protein genes of tobacco mosaic virus].

The present work demonstrated that RNA of different Tobacco mosaic virus strains (common; A14; Ni118, U2; wheat strain) are selectively cleaved by RNase H from E. coli in the presence of oligodeoxyribonucleotide d(TTTTTTTCCGG) complementary to the stretch of TMV RNA from the 842-nd to the 852-nd nucleotide from 3'-end, e. g. corresponding to the region adjacent to the 3'-terminus of origin of assemble of TMV. Such cleavage yields two main products. One of them--the short S-fragment--is the 3'-terminal part of TMV RNA. This conclusion is based on its ability to accept histidine and its molecular weight is about 0.28 X 10(6). The other main product of TMV RNA cleavage by RNase H is the L-fragment. L-fragment is the 5'-terminal moiety of TMV RNA, as follows from its ability to direct the in vitro synthesis of p110-130, which is known to be encoded in the 5'-terminal TMV RNA region.

Antigens, Viral↗

PubMed1984

5'-dephosphorylated 2',5'-adenylate trimer and its analogs. Inhibition of tobacco mosaic virus replication in tobacco mosaic virus-infected leaf discs, protoplasts, and intact tobacco plants.

The effect of the 5'-dephosphorylated 2',5'-adenylate trimer and its 2',5'-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linked immunosorbent assays. A structure-activity-metabolic stability-toxicity analysis of the 2',5'-adenylate trimer core molecule in TMV-infected Nicotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2' terminus (i.e. A-A-ara-A or A-A-Tu) inhibited replication of TMV. Modification of the 3'-hydroxyl group of the adenylate residues to 3-deoxyribose (i.e. the 2',5'-cordycepin trimer core) inhibited TMV replication better than the 2',5'-adenylate trimer core molecule. With enzyme-linked immunosorbent assays, there was complete inhibition of TMV replication by 200 nM 2',5'-adenylate trimer core for 60 h and by 200 nM 2',5'-cordycepin trimer core for 96 h. The amount of 2',5'-oligonucleotides associated with the leaves was determined using 2',5'-[3H]cordycepin trimer core; 1 X 10(-12) mol/cm2 of plant leaves inhibited TMV replication by 99%. No 2',5'-phosphodiesterase activity was detected in TMV-infected and noninfected leaf extracts. Therefore, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations, i.e. at 1000-fold lower concentration than that required in mammalian systems.

Adenosine↗

PubMed1994

Mapping helper virus functions for cucumber mosaic virus satellite RNA with pseudorecombinants derived from cucumber mosaic and tomato aspermy viruses.

P-TAV is a strain of tomato aspermy virus (TAV) able to efficiently support the systemic accumulation of some (i.e., B2-satRNA) but not of other (i.e., Ix-satRNA) strains of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) in both tobacco and in tomato. As reported for V-TAV, the failure to support the systemic accumulation of Ix-satRNA seems to be due to an inefficient support of its systemic movement. Pseudorecombinants obtained by the exchange of RNAs 1 + 2 between P-TAV and Trk7-CMV, an efficient helper for the systemic accumulation of Ix-satRNA, were assayed for their ability to support the accumulation of CMV-satRNAs in tobacco plants and protoplasts. Pseudorecombinants having RNAs 1 + 2 from CMV supported the systemic movement and accumulation of CMV-satRNA as efficiently as CMV, whereas pseudorecombinants having RNAs 1 + 2 from TAV supported the CMV-satRNA very poorly. Thus, the ability to support the systemic movement and accumulation of CMV-satRNA is determined primarily by RNAs 1 + 2 and not by RNA 3, which is presumed to encode movement functions in the cucumoviruses and only has a minor, modulating effect on the systemic accumulation of satRNA. This suggests that for systemic movement CMV-satRNA has to interact with (the gene products of) RNAs 1 and/or 2 or that these viral RNAs compete with the satRNA for interaction with the coat or other movement proteins.

Cucumovirus↗

PubMed1997

Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus.

cDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to express the jellyfish green fluorescent protein (GFP) in place of the 3a movement protein (MP) or coat protein (CP), as fusions to the N (GFP-3a) or C (3a-GFP) terminus of the MP or from a separate open reading frame as part of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect either Nicotiana tabacum or Nicotiana benthamiana systemically. Infection, as measured by confocal microscopy of GFP fluorescence, generally was limited to one to three epidermal cells at each inoculation site. Limited cell-to-cell movement, but not systemic movement, could be detected by complementation involving expression of MP and CP from two different RNA 3 constructs, each also expressing GFP. Infection involving RNA 3 expressing the GFP-3a fusion showed bright granules of variable size distributed predominantly and nonuniformly throughout the cytoplasm and, to a lesser extent, associated with the cell wall in single fluorescent cells, while infections expressing the 3a-GFP fusion showed bright, punctate fluorescence associated only with the cell wall. Infected cells expressing either 3a-GFP or free GFP showed a halo of less bright, fluorescent, neighboring cells, indicating limited movement of GFP. The initially infected cells also allowed movement of 10-kDa fluorescent dextran to the neighboring halo cells, while infection did not spread, suggesting different requirements for movement of either MP or dextran versus RNA.

Capsid↗

PubMed1998

Transgenically expressed cucumber mosaic virus RNA 1 simultaneously complements replication of cucumber mosaic virus RNAs 2 and 3 and confers resistance to systemic infection.

Tobacco plants transformed with a cDNA copy of RNA 1 of the Fny strain of cucumber mosaic virus (CMV) promoted the asymptomatic accumulation of inoculated viral RNAs 2 and 3, which could be detected in noninoculated leaves, suggesting that the transgene also permitted viral long-distance movement. Typical symptoms of infection appeared later and correlated with the appearance of viral RNA 1 regenerated from the transgenic mRNA. Although all R0-generation plants were susceptible to Fny-CMV, one line displaying variable susceptibility to the virus in R1-and R2-generations led to selected R3-generation lines with systemic resistance to Fny-CMV. In the inoculated leaves of resistant plants, a dramatic decrease in the accumulation of viral RNA 1 was observed, relative to susceptible plants. No viral RNAs were detected in noninoculated leaves of the resistant plants, but such leaves were susceptible to infection. Furthermore, these leaves could sustain replication of inoculated CMV RNAs 2 and 3, indicating that a complete transgene-silencing had not been induced. Although a transgene-mediated, CMV RNA 1-suppression occurred in the inoculated leaf of resistant plants, the absence of a complete systemically acquired silencing suggests the existence of additional interferences with viral infection that prevented systemic infection by viral RNAs 2 and 3.

Cucumovirus↗

PubMed2001

Crystallization of Brome mosaic virus and T = 1 Brome mosaic virus particles following a structural transition.

Brome mosaic virus (BMV), a T = 3 icosahedral plant virus, can be dissociated into coat protein subunits and subunit oligomers at pH 7.5 in the presence of concentrated salts. We have found that during the course of this treatment the coat protein subunits are cleaved, presumably by plant cell proteases still present in the preparation, between amino acids 35 and 36. The truncated protein subunits will then reorganize into T = 1 icosahedral particles and can be crystallized from sodium malonate. Quasi elastic light scattering and atomic force microscopy results suggest that the transition from T = 3 to T = 1 particles can occur by separate pathways, dissociation into coat protein subunits and oligomers and reassembly into T = 1 particles, or direct condensation of the T = 3 virions to T = 1 particles with the shedding of hexameric capsomeres. The latter process has been directly visualized using atomic force microscopy. Native T = 3 virions have been crystallized in several different crystal forms, but neither a rhombohedral form nor either of two orthorhombic forms diffract beyond about 3.4 A. Tetragonal crystals of the T = 1 particles, however, diffract to at least 2.5 A resolution. Evidence suggests that the T = 1 particles are more structurally uniform and ordered than are native T = 3 virions. A variety of anomalous virus particles having diverse sizes have been visualized in preparations of BMV used for crystallization. In some cases these aberrant particles are incorporated into growing crystals where they are frequently responsible for defect formation.

Bromovirus↗