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K L Perry

Publications and source records attributed to K L Perry.

At least 19 recordsLinked to original sources
PubMed2004

A new potato virus in a new lineage of picorna-like viruses.

On constructing a cDNA library for potato, 'contaminating' sequences with a significant identity to Apple latent spherical virus (ALSV) were found. Determination of the remaining genome sequence indicated the presence of a bipartite virus with an RNA1 and 2 of 7034 and 3315 nucleotides, respectively, excluding a poly(A)tail. RNA1 encodes a single polyprotein (233 kDa) and shares highest amino acid identity with ALSV at 65%. Conserved amino acid motifs typical for helicase, protease and RNA-dependent polymerase (RdRp) functions are present. RNA2 encodes a single polyprotein (106 kDa) with amino acid identities to the flat apple isolate of Cherry rasp leaf virus (CRLV-FA) (97%) and ALSV (70%), suggesting this is a potato strain of CRLV (CRLV-pot). Phylogenetic analysis using the RdRp region shows that this virus falls within a group separate from the Comoviridae that includes members of the Sequiviridae and the taxonomically unassigned viruses ALSV, Strawberry mottle virus, Satsuma dwarf virus and Navel orange infectious mottling virus. Other regions of the genome have highest identities with both plant and animal infecting members of the picorna-like virus superfamily. The evolutionary context of CRLV-pot and related viruses is discussed. Similar viral sequences from an EST library of peppermint are also analysed.

5' Untranslated Regions↗
PubMed2000

Cucumber mosaic virus mutants with altered physical properties and defective in aphid vector transmission.

Two mutant strains of cucumber mosaic virus (CMV) were investigated with respect to virion stability and molecular determinants of aphid vector transmission. The mutant 2A1-MT-60x, derived from the mechanically passaged wild type 2A1-AT, is poorly transmissible by the aphid Aphis gossypii and not transmissible by the aphid Myzus persicae, whereas the wild type virus is transmissible by both aphid species. The mutant phenotype was shown to be conferred by a single encoded amino acid change of alanine to threonine at position 162 of the coat protein (CP). Modifying the mutant CP gene to encode the wild type sequence (alanine) at position 162 restored aphid transmission. To test for a correspondence between changes in the physical stability of virions and defects in aphid transmission, a urea disruption assay was developed. Virions of aphid-transmissible strains 2A1-AT and CMV-Fny were stable with treatments of up to between 3 and 4 M urea. In this assay mutant viruses 2A1-MT-60x and CMV-M were less stable, as they were completely disrupted at urea concentrations of 2 and 1 M urea, respectively. The mutant 2A1-MT-60x also accumulated at a reduced level in infected squash relative to the wild type virus. These studies suggest that a primary factor in the loss of aphid transmissibility of some strains of CMV is a reduction in virion stability.

Amino Acid Substitution↗
PubMed2000

The structure of cucumber mosaic virus and comparison to cowpea chlorotic mottle virus.

The structure of cucumber mosaic virus (CMV; strain Fny) has been determined to a 3.2-A resolution using X-ray crystallography. Despite the fact that CMV has only 19% capsid protein sequence identity (34% similarity) to cowpea chlorotic mottle virus (CCMV), the core structures of these two members of the Bromoviridae family are highly homologous. As suggested by a previous low-resolution structural study, the 305-A diameter (maximum) of CMV is approximately 12 A larger than that of CCMV. In CCMV, the structures of the A, B, and C subunits are nearly identical except in their N termini. In contrast, the structures of two loops in subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues longer than the analogous regions in CCMV. Unlike that of CCMV, the capsid of CMV does not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due to the fact that the N termini of the B and C subunits form a unique bundle of six amphipathic helices oriented down into the virion core at the threefold axes. In addition, while CCMV has a cluster of aspartic acid residues at the quasi-threefold axis that are proposed to bind metal in a pH-dependent manner, this cluster is replaced by complementing acids and bases in CMV. Finally, this structure clearly demonstrates that the residues important for aphid transmission lie at the outermost portion of the betaH-betaI loop and yields details of the portions of the virus that are hypothesized to mediate binding to aphid mouthparts.

Amino Acid Sequence↗
PubMed1998

Amino acid changes in the coat protein of cucumber mosaic virus differentially affect transmission by the aphids Myzus persicae and Aphis gossypii.

Cucumber mosaic virus (CMV) transmission by two aphid species was investigated using strains of the virus with modified coat proteins. Chimeric viral RNA 3 cDNA constructs were designed with coat protein-encoding sequences from the efficiently aphid-transmitted Fny-CMV and the very poorly aphid-transmitted M-CMV. Different regions of the viral coat protein variably influenced transmission; the magnitude of effects depended on the aphid species used as vector. Five amino acid changes in the coat protein of M-CMV (positions 25, 129, 162, 168, and 214) were required to restore efficient transmission by Myzus persicae. This contrasts with previously demonstrated, more limited changes required for restoration of transmission by Aphis gossypii. A construct with modified amino acids 129, 162, and 168 was efficiently transmitted by A. gossypii, while remaining poorly transmissible by M. persicae. Transmission of Fny-CMV by single aphids of A. gossypii and M. persicae was 54.0% and 27.9%, respectively. Single coat protein amino acid changes in Fny-CMV dramatically reduced transmission by M. persicae, while showing little effect on transmission by A. gossypii.

Animals↗
PubMed1998

Identification of epitopes in cucumber mosaic virus using a phage-displayed random peptide library.

Antigenic sites in the cucumber mosaic virus (CMV) coat protein (CP) have been identified using a polyclonal antiserum prepared against glutaraldehyde-fixed virions. Antibodies were used to screen a random peptide library of heptamers displayed on the surface of a bacteriophage. Eight of 36 (22%) sequenced phage clones had inserts resembling a putative virion surface domain of the CMV CP. This region has the sequence LETDEL, corresponding to amino acids 194-199 in the Fny-CMV CP. The binding of phage clones to Fny-CMV antiserum was inhibited by a synthetic peptide representing this region. Six of 36 (17%) phage clones contained sequences corresponding to a C-terminal sequence in the Fny-CMV CP, which is thought to be internal in assembled virions. This sequence, EHQRIPTSGV, represents amino acids 206-215 and all but the P residue were observed in at least one clone. Four of 36 (11%) sequenced phage clones carried sequences that matched a portion of the sequence RLLLPDSV, corresponding to amino acids 89-96 in the Fny-CMV CP. This region was also identified as the antigenic site recognized by a monoclonal antibody (MAb23C10E4). Eleven percent of the phage (4 of 36) contained sequences matching at least three amino acids of the N-terminal region in the CMV CP. The positions of the antigenic sites seen in this study are consistent with a predicted structure for the CMV CP.

Antibodies, Monoclonal↗
PubMed1994

Specificity of replicase-mediated resistance to cucumber mosaic virus.

Plants transformed with a nucleotide sequence coding for a truncated RNA 2 replicase gene of the subgroup I strain of cucumber mosaic virus, Fny-CMV, are resistant to cucumber mosaic disease. Two resistant lines representing independent transformations in the original study have been propagated, and their progeny have been examined. Resistance to Fny-CMV was genetically integrated and was retained in the R4 generation. Fourteen subgroup I CMV strains, 6 strains uncharacterized as to subgroup, and 4 subgroup II CMV strains, as well as potato virus X, tobacco mosaic virus, tomato aspermy virus, tobacco etch virus, and peanut stunt virus were inoculated mechanically to R2 generation plants of these two lines. Resistance was found to 12 subgroup I CMV strains and to all of the subgroup-uncharacterized strains. No resistance was found to subgroup II CMV strains nor to the other viruses listed above. However, the two plant lines behaved differently in response to 3 subgroup I strains, K-CMV, PRC-CMV, and MB-CMV, where one line (5) was more resistant than the other (2). RNase protection analyses indicated that these 3 strains vary more in sequence from Fny-CMV than do the other subgroup I strains tested. Resistance was also shown when aphids were used to inoculate plants with either of 2 subgroup I CMV strains; no resistance was observed when aphids were used to transmit a subgroup II CMV strain. Inoculation of transgenic plants with pseudorecombinants produced with the RNAs derived from Fny-CMV to which the plants are resistant, and LS-CMV, a subgroup II strain to which they are susceptible, indicated that the resistance-breaking capacity of LS-CMV is a function of RNA 2.

Animals↗
PubMed1994

Mapping determinants in cucumber mosaic virus for transmission by Aphis gossypii.

Determinants have been mapped in the coat protein of cucumber mosaic virus (CMV) which influence transmission by the aphid Aphis gossypii. Chimeric RNA 3s were constructed which contain sequences from the efficiently aphid-transmitted strain Fny-CMV and the very poorly aphid-transmissible M-CMV. Analyses of chimeric viruses revealed that two regions in the coat protein were critical for transmission by aphids. The coat proteins of Fny- and M-CMV were previously shown to differ at eight amino acid positions. Amino acid changes were introduced into regions of the coat protein gene of M-CMV shown to be involved in transmission and two amino acid changes were demonstrated as essential for the restoration of aphid transmissibility. Nonconservative changes at positions 129 (Leu to Pro) and 162 (Thr to Ala) modified the coat protein to resemble Fny-CMV at these positions and restored transmissibility by A. gossypii. A change at amino acid position 168 (Cys to Tyr) may influence the efficiency of aphid transmission, but is not essential. A single amino acid change at position 129 (Leu to Pro) did not restore aphid transmissibility to M-CMV. The findings are discussed in relation to the nonpersistent transmission of plant viruses.

Animals↗
PubMed1994

Case II diffusion in the PVC and acetone system.

This paper describes how nuclear magnetic resonance (NMR) has been used to study case II diffusion of acetone into PVC. NMR is supported by Ion Beam Nuclear Reaction Analysis and Gravimetric Analysis. A series of experiments with different PVC exposure times to acetone vapour have shown the observable physical characteristics of case II diffusion. Experiments in the temperature range 20-50 degrees C have yielded the temperature dependence of the front velocity and the T2 values of the acetone and PVC components.

Acetone↗
PubMed1992

Insect-mediated transmission of mixed and reassorted cucumovirus genomic RNAs.

Transmissions of virus using the aphid Myzus persicae were performed using plants co-infected with two cucumoviruses, tomato aspermy virus (V-TAV) and cucumber mosaic virus (M-CMV). Five of the aphid-transmitted progeny viruses (3.7%) induced symptoms distinct from those induced by either parental virus. Northern blot hybridization analysis of encapsidated RNAs from these novel progeny demonstrated that all of the RNA profiles were characteristic of pseudo-recombinants, i.e. viruses with reassorted genomic RNAs. The two larger RNAs, 1 and 2, originated from V-TAV, whereas RNA 3 was derived from M-CMV. A more sensitive RNase protection assay analysis of both unencapsidated and encapsidated RNAs revealed the presence of minor populations of V-TAV-derived RNA 3 in all of these novel progeny, and of M-CMV-derived RNA 1 (and presumably RNA 2) in one of the progeny. A bias against the encapsidation of the minor populations of RNAs by the M-CMV coat protein was observed, suggesting that there is specificity or competition with regard to the encapsidation of cucumoviral RNAs in vivo. This study demonstrates that insect vectors can mediate the establishment of pseudorecombinants with mixed populations of RNA 3.

Animals↗
PubMed1990

Transcription of tomato ribosomal DNA and the organization of the intergenic spacer.

The organization of the intergenic spacer of a 9.04 kb tomato ribosomal RNA gene (rDNA) was determined. The 3258 bp spacer contains two major repeat elements enclosing a region which includes 351 bp of an 81.8% A --T rich sequence. A block of nine 53 bp repeats begins 388 bp downstream from the 3' end of the 25S rRNA. The A--T rich domain is followed by a block of six 141 bp repeats terminating 818 bp upstream from the 5' end of the 18S rRNA. Major pre-rRNAs of 7.6 and 6.5 kb were observed by Northern hybridization analysis. The 5' termini of these RNAs were identified through combined S1 nuclease and primer extension analyses. The 7.6 kb RNA is likely to be the primary transcript; its 5' terminus lies within a sequence motif. TATA(R)TA(N)GGG, conserved at the termini of transcripts mapped in three other plant species. The 6.5 kb RNA is interpreted as a 5' end processed transcript derived from the 7.6 kb RNA. Comparative analysis of transcribed sequences revealed a 25 bp domain of the intergenic spacer which is relatively conserved among five plant species. The conservation of spacer sequences in plants is in contrast to the extensive sequence divergence of the intergenic spacer in other non-plant systems and suggests a conserved function directed by these sequences.

Base Sequence↗
PubMed1989

Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes.

Animals↗
PubMed1989

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.

Animals↗
PubMed1987

Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader.

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.

Animals↗
PubMed1986

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.

Cloning, Molecular↗
PubMed1985

umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology.

The products of the Escherichia coli umuDC operon and its plasmid-borne analog, mucAB, are required for mutagenesis caused by UV light and by many chemicals. We have determined the nucleotide sequences of umuDC and mucAB and present comparisons of these sequences. The two operons are 52% homologous at the nucleotide level. Open reading frames corresponding in position and size to the umu and muc genes have been identified. The reading frames of umuD and umuC overlap by 1 base pair, and the reading frames of mucA and mucB overlap by 13 base pairs. The predicted amino acid sequences of the UmuD and MucA proteins are 41% homologous; those of the UmuC and MucB proteins are 55% homologous. Considerable homology has also been detected between UmuD, MucA, and the COOH-terminal domains of the LexA repressor and the repressors of phage lambda, 434, and P22. Complementation analyses reveal that MucA protein cannot substitute for UmuD in a umuD- umuC+ host and that MucB protein cannot substitute for UmuC in a umuD+ umuC- host. Potential regulatory sequences have been identified in umuDC and mucAB.

Amino Acid Sequence↗
PubMed1984

Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.

Base Sequence↗