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J M Kaper

Publications and source records attributed to J M Kaper.

At least 37 records · Page 2Linked to original sources
PubMed1988

Site-directed mutagenesis of potential protein-coding regions in expressible cloned cDNAs of cucumber mosaic viral satellites.

Site-directed mutagenesis was used to alter potential protein-coding regions (open reading frames or ORFs) within the cloned, expressible cDNAs of two satellites of cucumber mosaic virus (CMV), S-CARNA 5 and D-CARNA 5. RNA transcripts synthesized in vitro from the mutant and wild-type satellite cDNAs were tested for biological activity on tomato plants by coinfection with CMV RNAs 1, 2, and 3, and progeny CARNA 5s generated from such transcripts were isolated and sequenced. Two mutants of S-CARNA 5 were constructed in attempts to test whether the ORF (ORF IIB, beginning at nucleotide 135) responsible for synthesis in vitro of two small proteins (M. J. Avila-Rincon, C. W. Collmer, and J. M. Kaper (1986). Virology 152, 455-458) could be eliminated without loss of the satellite's biological activity. Biological tests with mutant transcripts that either lacked the AUG initiation codon or contained a premature translation termination codon were foiled by instability and/or reversion in the progeny. With progeny CARNA 5 of the former mutant, one of two altered nucleotides reverted, thus restoring the AUG but not the nucleotide immediately preceding it. In contrast, a mutation in the necrogenic D-CARNA 5 which altered the initiation codon of ORF I (nucleotides 11-94), whose predicted amino acid sequence is conserved in all necrogenic CMV satellites sequenced to date, was stable in biological testing and did not destroy necrogenicity. This was shown by nucleotide sequencing of the progeny CARNA 5 from necrotic test plants and by its direct comparison with wild-type D-CARNA 5 progeny in a tomato necrosis dilution assay. This experiment provides convincing evidence that a possible protein product of ORF I is not involved in the induction of tomato necrosis by D-CARNA 5.

Codon↗
PubMed1986

In vitro translation of cucumoviral satellites. II. CARNA 5 from cucumber mosaic virus strain S and SP6 transcripts of cloned (S)CARNA 5 cDNA produce electrophoretically comigrating protein products.

This report shows that (S)CARNA 5, the satellite of cucumber mosaic virus (CMV) strain S, directs the synthesis of two small proteins in a wheat germ cell-free translation system. The two proteins are produced by the translation of both (S)CARNA 5 purified from CMV virions and the (+) strand purified from double-stranded (S)CARNA 5. In addition, two protein products that comigrate electrophoretically with those above are produced when transcripts synthesized in vitro from full-length (S)CARNA 5 cDNA cloned in the plasmid pSP65 are substituted for (S)CARNA 5 in the translation system. The two proteins therefore must derive from a single (S)CARNA 5 sequence.

Cloning, Molecular↗
PubMed1986

Infectious RNA transcripts from cloned cDNAs of cucumber mosaic viral satellites.

Complete cDNA copies of two variants of CARNA 5, the satellite of cucumber mosaic virus, have been cloned in the transcription vector pPM1. These two naturally-occurring CARNA 5s are capable and incapable, respectively, of inducing a lethal necrotic disease of tomato upon coinoculation with the genomic RNAs 1, 2, and 3 of cucumber mosaic virus. Uncapped transcripts synthesized in vitro from the two linearized, recombinant plasmids are infectious and each induces the appropriate symptomatology upon coinfection with cucumber mosaic viral RNAs on tomato plants. Progeny CARNA 5s isolated from such infected plants correspond to their natural CARNA 5 counterparts.

Cloning, Molecular↗
PubMed1985

Nucleotide sequence of the satellite of peanut stunt virus reveals structural homologies with viroids and certain nuclear and mitochondrial introns.

Peanut stunt virus-associated RNA 5 (PARNA 5), the satellite of a plant cucumovirus, is a linear RNA of 393 nucleotides with a 5' cap and a 3' hydroxyl group. Determination of its nucleotide sequence has revealed two consecutive open reading frames that together extend most of its length. Sequences at the 5' and 3' ends are homologous with those of the satellite of the related cucumber mosaic virus, and the double-stranded forms of both satellites contain an unpaired guanosine at the 3' end of the minus strand. However, little other homology exists between the two satellites. In contrast, PARNA 5 has several regions of 90% sequence homology with various plant viroids, including sequences of the conserved central region of most viroids. Such homologies suggest a common origin with viroids coupled with specific adaptation as a linear RNA. The presence within PARNA 5 of conserved intron sequences essential to proper RNA processing suggests a possible origin from plant introns and/or involvement of such sequences in the processing of PARNA 5 multimers to monomers at some stage of replication.

Base Sequence↗
PubMed1984

Replication of peanut stunt virus and its associated RNA 5 in cowpea protoplasts.

Peanut stunt virus (PSV) RNA containing PSV-associated RNA 5 (PARNA 5) was used as the inoculum in the successful infection of cowpea protoplasts. Total nucleic acid extracts of protoplast samples at different times after inoculation were analyzed for the presence of PSV genomic RNAs and PARNA 5 using glyoxal denaturation, agarose gel electrophoresis, blotting to nitrocellulose, and hybridization to specific probes. It appears that (+)-stranded PSV genomic RNAs are synthesized up to 36 hr after inoculation after which their synthesis levels off, whereas PARNA 5 synthesis continues much later during infection. Oligomers of PARNA 5 were found in the infected protoplasts, in double-stranded RNA preparations from PSV-infected tissues, and in single-stranded PSV-RNA preparations isolated from purified virus. However, we were unable to demonstrate the presence of circular PARNA 5 molecules in infected protoplasts or tissues. These results leave open the question whether PARNA 5 is replicated via a rolling circle type replication mechanism, as proposed for viroids and tobacco ringspot virus satellite, or via a virus-like replication mechanism, as certain structural features of PARNA 5 would indicate. It is not impossible that both types of mechanisms are operative at different phases of PARNA 5 replication.

Arachis↗
PubMed1983

Separation of the complementary strands of double-stranded cucumber mosaic virus-associated RNA 5 and peanut stunt virus-associated RNA 5.

The double-stranded forms of CARNA 5 and PARNA 5, viral satellites of the cucumovirus group, after denaturation and polyacrylamide gel electrophoresis under appropriate conditions allow their complementary strands to be separated and fractionated. These fractionated (+) and (-) strands are free of contaminating viral RNA fragments. They serve as templates for the preparation of cDNAs of opposite polarity, which are used to probe the viral RNA-dependent replication of CARNA 5 and PARNA 5.

Arachis↗
PubMed1982

Stiffness of viroids and viroid-like RNA in solution.

The sedimentation coefficients of the potato spindle tuber viroid, four viroid-like RNAs from cadang-cadang-disease, circular RNA from velvet tobacco mottle virus, circular RNA from Solanum nodiflorum mottle virus and double stranded RNA5 from cucumber mosaic virus were measured in the analytical ultracentrifuge. The numbers of nucleotides of the RNA species varied between 246 and 670. The hydrodynamic models of rigid rods and flexible cylinders were applied for the interpretation of the sedimentation coefficients. Double-stranded RNA5 from cucumber mosaic virus with 335 basepairs fits the model of a rigid rod with an hydrated diameter of 29 A. Potato spindle tuber viroid and the four viroid-like RNA species of cadang-cadang-disease form a homologous series of flexible cylinders with a Kuhn's statistical length lambda-1 of 600 A. The circular RNA from the two viruses mentioned above are more flexibel than the viroids and viroid-like RNAs. The hydrodynamic interpretation is in accordance with thermodynamic data and secondary structure models. In two of the RNAs from cadang-cadang, cruciform structures would also be possible on the basis of the nucleotide sequence. The hydrodynamic data, however, favour clearly the extended structure over the cruciform.

Genes, Viral↗
PubMed1979

Cucumber mosaic virus-associated RNA 5. VI. Characterization and denaturation-renaturation behavior of the double-stranded form.

The double-stranded form of cucumber mosaic virus-associated RNA 5 has been purified and further characterized. Its molecular weight determined by sedimentation equilibrium is 2.15 . 10(5). The buoyant density calculated from its symmetrical distribution in Cs2SO4, following isopycnic ultracentrifugation, is 1.615 g/cm3. The sedimentation rate of double-stranded cucumber mosaic virus-associated RNA 5 is slightly greater than that of cucumber mosaic virus-associated RNA 5; its electrophoretic mobility in polyacrylamide gel (2.4%) is less than that of cucumber mosaic virus-associated RNA 5. By the above standards the double-stranded cucumber mosaic virus-associated RNA 5 preparations used were found to be nomogeneous in size as well as density. Thermal denaturation monitored by means of ultraviolet light absorption produced multitransitional denaturation profiles. The average melting temperature (Tm) was 88 degrees C in 0.1 x SSC. Monotransitional denaturation profiles and slightly higher Tm values were obtained when resistance against ribonuclease digestion was measured. These denaturation experiments and other propertied led to the conclusion that double-stranded cucumber mosaic virus-associated RNA 5 and the double-stranded form of peanut stunt virus-associated RNA 5 are small double-stranded nucleic acids with several homostable base-pair regions, characterized by distinct G + C contents and Tm values.

Kinetics↗
PubMed1978

Studies of virus structure by laser-Raman spectroscopy. Turnip yellow mosaic virus and capsids.

Laser-Raman spectroscopy of the turnip yellow mosaic virus (TYMV) and its capsid indicate the following features of the structure and assembly of the virion. The secondary structure of coat-protein molecules in TYMV is comprised of 9 +/- 5% alpha-helix, 43 +/- 6% beta-sheet, and 48 +/- 6% irregular conformation and is not altered by the removal of the RNA from the capsid. Introduction of as many as 200 chain scissions per RNA molecule also does not affect the overall secondary structure of the encapsulated RNA, which is 77 +/- 5% in the A-helix form. Tryptophan and cysteine residues of the coat protein appear to be in contact with the solvent, while only one of three tyrosines per coat protein is available for hydrogen bonding of its p-hydroxyl group with H2O molecules. Both cytosine and adenine residues of TYMV RNA are protonated in substantial numbers near pH 4.5, suggesting elevation of their respective pKa values within the virion. The Raman data are consistent with chemical evidence favoring interaction between protonated bases of RNA and amino acid side chains of coat protein in TYMV.

Capsid↗
PubMed1978

Isolation of viral double-stranded RNAs using a LiCl fractionation procedure.

A general procedure for the isolation of virus-specific double-stranded RNA (ds-RNA) is discribed. The procedure is based on the differential solubility of different types of nucleic acids in LiCl. Principal advantages over conventional methods are simplicity, avoidance of enzymatic treatment, and relatively good yields of undegraded ds-RNA while permitting separation of several main groups of cellular and viral nucleic acids from the same batch of tissue. The method has been successfully applied in tissues infected by several representative plant RNA viruses. The virus-specific ds-RNAs obtained have been identified by their resistance to ribonuclease and comparison of their electrophoretic mobilities with those of the corresponding single-stranded RNA (ss-RNA) in polyacrylamide gels. The molecular weights of the ds-RNAs of tobacco mosaic virus, turnip yellow mosaic virus, alfalfa mosaic virus, and peanut stunt virus fit the curved log molecular weight-migration relationship constructed from a set of known marker ds-RNAs.

Chlorides↗