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U Melcher

Publications and source records attributed to U Melcher.

At least 37 records · Page 2Linked to original sources
PubMed1993

HIV-1 proteinase as structural model of intercellular transport proteins of plant viruses.

Intracellular movement of viral infections of plants requires a virus-encoded protein. Alignment of amino acid sequences of central conserved regions of such proteins produced a sequence profile that resembled that of lentiviral proteinases. The known three-dimensional structure of the proteinase encoded by the human immunodeficiency virus-1 (HIV-1) may serve as a model for the three-dimensional structure of the central region of the plant viral proteins. Secondary structures predicted for the plant viral proteins from their amino acid sequences correlate well with those predicted from a proteinase model. In addition, the positions of temperature-sensitive and resistance-breaking mutations in the intercellular transport protein of tobacco mosaic virus are consistent with a structural similarity between the plant viral proteins and the lentiviral proteinases. In a suggested model, the dimeric proteinase-similar domain serves as tether for the attachment of N- and C-terminal domains. The C-terminal domain may be an RNA-binding domain. The similarity was used to assign intercellular transport function to a previously unidentified coding region of the genomes of bacilliform DNA viruses.

Amino Acid Sequence↗
PubMed1993

A method for the isolation of nuclear DNA from cotton (Gossypium) leaves.

Cotton leaves contain high levels of polyphenolic compounds that irreversibly interact with proteins and nucleic acids during DNA isolation. A procedure to isolate nuclear DNA from cotton (Gossypium hirsutum L.) has been developed. The method is based on the rapid initial isolation of nuclei in a glucose medium designed to stabilize nuclear structure and composition while preventing covalent interactions with polyphenolics. The resulting DNA is high in yield and purity and is suitable for Southern-blot hybridization analysis and restriction fragment length polymorphism analysis.

Blotting, Southern↗
PubMed1993

In planta deletion of DNA inserts from the large intergenic region of cauliflower mosaic virus DNA.

RNA splicing and copy-choice recombination lead to deletions in the DNA of cauliflower mosaic virus (CaMV). To assess the relative importance of these mechanisms of nucleotide sequence change, free of constraints imposed by the "relay race" mode of translation of CaMV RNA, we examined the stability of inserts in the large intergenic region. The insertions had, in various combinations, splicing signals and directly repeated sequences that could facilitate deletion. Most modified DNAs were infectious. Viral DNA recovered from infected plants was analyzed by restriction and, in some cases, cloned for nucleotide sequencing to determine deletion endpoints. Deletions from a DNA containing introduced splicing signals occurred primarily at direct repeats, although deletion apparently by splicing was also detected. Both types of deletion were also observed with insertions containing a 5' splice donor but no known functional 3' splice acceptor. One DNA, whose insertion lacked splicing signals and short repeated sequences, was stable in one of two plants infected. Total insert deletions were bounded by repeats or pseudorepeats, while partial insert deletions apparently occurred by splicing. The results suggest that the two mechanisms for deletion of nucleotides are equally important in the evolution of caulimoviral nucleotide sequences.

Base Sequence↗
PubMed1993

Coat protein of cauliflower mosaic virus binds to ssDNA.

Alkali-denatured cauliflower mosaic virus (CaMV) virions incorporated ssDNA, added exogenously, into multimolecular complexes during dialysis against a neutral buffer. CaMV coat protein binding to tracer DNA, assessed by gel electrophoresis and autoradiography, was highly cooperative as judged by the absence of intermediate-sized complexes. The incorporation of labelled BglII fragments of a plasmid containing CaMV DNA into complexes was prevented by the presence of 0.16 g/l unlabelled calf thymus DNA. Lower concentrations of competitor DNA allowed binding of some BglII fragments although preventing the binding of others. The self-annealing poly(dI-dC) was much less efficient than calf thymus DNA in preventing the incorporation of fragments into complexes, suggesting a binding preference for ss- over dsDNA. In addition, dsDNA, minimally cross-linked to prevent strand separation, was bound only weakly. End-labelled ssDNA fragments in complexes were partially protected against DNase I. The nucleic acid-binding activity of CaMV coat protein may be responsible for the organization of replication complexes, the precursors to virion particles.

Binding, Competitive↗
PubMed1992

Diverse mechanisms of plant resistance to cauliflower mosaic virus revealed by leaf skeleton hybridization.

Plants not hosts for cauliflower mosaic virus (CaMV) may prevent systemic CaMV infection by interfering with dissemination of infection through the plant or by preventing viral replication and maturation. Leaf skeleton hybridization allows distinction between these two barriers. The technique assesses the spatial distribution of CaMV in an inoculated leaf by hybridization of a skeleton of the leaf with a CaMV DNA probe. Leaves or leaflets of soybean, cucumber, peanut, tomato, lettuce, spinach, pepper, onion, wheat, maize and barley, inoculated with CaMV DNA or CaMV virions were processed for leaf skeleton hybridization either immediately after inoculation or two weeks thereafter. Autoradiographic images of soybean and cucumber skeletons had many dark spots suggesting that CaMV DNA replication and local spread had occurred. Images of onion leaf skeletons prepared two weeks after inoculation with CaMV DNA had fewer spots. To test whether these spots resulted from CaMV replication, DNA was extracted from inoculated onion leaves and analyzed by electrophoresis, blotting and hybridization. Molecules recovered two weeks after inoculation resembled those inoculated, indicating absence of replication. For the other species, we found no evidence of local spread of CaMV infections. Thus, many plant species resist systemic CaMV infection by preventing replication or local spread of CaMV, while others solely prevent systemic movement of infection.

Blotting, Southern↗
PubMed1990

Recombination sites in cauliflower mosaic virus DNAs: implications for mechanisms of recombination.

Pairs of mutant cauliflower mosaic virus (CaMV) DNAs readily recombine in plants. Five plasmid clones of CaMV DNAs resulting from infection of turnips with pairs of mutant DNAs from DNAs resulting from infection of turnips with pairs of mutant DNAs from different isolates were obtained. Restriction analysis and nucleotide sequencing identified deletions in two cloned recombinants, VR1249 and VR244B. The sequence missing in the former was consistent with its deletion by splicing of an RNA intermediate. These DNAs were not infectious in turnips. VR1243, VR244A, and VR246 induced in turnips disease symptoms that were mixtures of those produced by the parental isolates. Junctions between sequences of the parental isolates were identified by restriction fragment analysis. Three cloned chimeras resulted from multiple recombination events. Nucleotide sequencing identified more precisely the junctions in the five cloned chimeras and in three chimeras previously characterized. Consistent with a model in which reverse transcription plays a major role in generating recombinants, six chimeras had junctions at or near the site for initiation of DNA(-) strand synthesis, three had junctions near the initiation site of 35 S RNA transcription, and one junction was found near the initiation site of 19 S mRNA transcription. Junctions were also found in regions not bearing any obvious relation to DNA (-) strand synthesis by reverse transcription, suggesting that recombination of double-stranded DNAs may also generate CaMV DNA recombinants.

Base Sequence↗
PubMed1990

Similarities between putative transport proteins of plant viruses.

The nucleic acids of many plant viruses encode proteins with one or more of the following properties: an Mr of approximately 30,000, localization in the cell wall of the infected plant and a demonstrated role in cell-to-cell transport of infection. A progressive alignment strategy, aligning first those sequences known to be similar, and then aligning the resulting groups of sequences, was used to examine further the relatedness of the amino acid sequences of putative transport proteins of caulimoviruses, of proteins similar to the putative transport protein of alfalfa mosaic virus (A1MV) and of those similar to the tobacco mosaic virus (TMV) 30K protein. The strategy first identified regions in which multiple dipeptides of one group were similar to those of another group. The regions of similarity were brought into alignment by the conservative introduction of gaps. The positions of the introduction of gaps were adjusted to optimize similarity. Statistical significances of the resulting alignments, determined both by comparison with shuffled amino acid sequences and with the sequence alignment off-set by 1 to 15 residues in each direction, suggest that the amino acid sequences of the three groups of viruses are distantly related. Nevertheless, significant relationships between members of the caulimoviral group of sequences and members of each of the A1MV-like and TMV-like groups were found. These relationships and the analysis of the number of insertions/deletions between present sequences and a hypothetical common ancestor suggest that the sequences of the caulimoviral proteins are less diverged from the ancestor than either the A1MV-like or TMV-like proteins. The alignment identified common regions of predicted secondary structure and regions of similar hydropathy, regions possibly crucial for proper functioning of the proteins.

Amino Acid Sequence↗
PubMed1988

A readable and space-efficient DNA sequence representation: application to caulimoviral DNAs.

A new representation of nucleotide sequence information has been devised to allow the clear presentation of large amounts of sequence information in a limited amount of space. Nucleotide sequences of DNA are represented by squares in three vertically aligned positions for each nucleotide. The squares represent, from top to bottom: a purine, a guanine or cytosine and a pyrimidine. The representation provides a simultaneous representation of both strands of double-stranded DNA and can be easily scanned visually for a variety of sequence features. Biologically important features of the DNA sequence near the origin of SV40 replication are easily recognized. The representation was also used to identify possibly base-paired stems in caulimoviral transcripts that may be of significance for viral replication.

Base Sequence↗
PubMed1987

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton.

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.

Journal Article↗
PubMed1986

Infectious and non-infectious mutants of cauliflower mosaic virus DNA.

Mutants of cauliflower mosaic virus (CaMV), generated in vitro by modification of recombinant DNA plasmids containing the viral genome, either retained the ability to induce disease symptoms on turnip plants, produced less severe symptoms or failed to induce symptoms. Wild-type symptoms were produced by a variant CaMV DNA of the Cabbage S isolate that had 4 bp in open reading frame (ORF) III replaced with a 16 bp sequence. Less severe symptoms, due to a delay in symptom appearance relative to inoculation with wild-type DNA, were induced by a mutant with a frameshift mutation in ORF II (pSA103). CaMV DNA, recovered from plants infected with pSA103, contained a second mutation which restored the original translation reading frame. Nucleic acid hybridization to 'squishes' of leaf tissue from plants that had been inoculated with mutant DNAs that included DNAs modified in each of the six major ORFs of CaMV DNA revealed that only those plants that appeared diseased had detectable CaMV nucleic acid in uninoculated leaves. Replicated CaMV DNA was also not detected in non-encapsidated and virion DNA fractions from inoculated leaves of non-diseased plants.

Base Sequence↗
PubMed1983

Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies.

In a new combination of techniques for ribosomal RNA hybridization, complementary DNA is synthesized on 25S ribosomal RNA fragments generated by mild alkali treatment, by the enzymatic addition of polyadenylic acid tails, hybridization of these tails with oligo deoxyribosylthymine, and reverse transcription in the presence of tritiated TTP. Hybridization reactions are performed in solution. Heteroduplexes are collected on diethylaminoethylcellulose filter discs after treatment with S1 nuclease. The problems presented by secondary rRNA structure are avoided by denaturation before reverse transcription and before hybridization. The high percentage of duplex formation (78-87%), the low standard deviation of relative binding (averaging +/- 1.30% relative binding), and small differences in reciprocal hybridizations (1.71-5.18% relative binding), as well as the elimination of complications resulting from differences in the number of rRNA cistrons in nuclear DNA, make this method preferable to the membrane-filter technique commonly used in phylogenetic classifications based on the homology of large rRNAs.

DNA↗
PubMed1979

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.

Animals↗
PubMed1979

In Vitro Synthesis of a Precursor to the Methionine-rich Polypeptide of the Zein Fraction of Corn.

The messenger ribonucleic acid fraction isolated from a protein bodyenriched fraction of developing corn (Zea mays L.) endosperm stimulated the incorporation of radioactive amino acids into at least five polypeptides when added to a wheat germ extract capable of protein synthesis. Of these, the two major polypeptides formed with messenger from freshly frozen corn were identified as precursors to zein A and B, the two major polypeptides of the prolamine fraction of corn meal (21,600 and 19,600 molecular weight). The identification was based on the relative incorporations of radioactive leucine, lysine, and methionine, and the susceptibility of the zein A precursor, but not the zein B precursor to cleavage with cyanogen bromide. Using extracts from stored frozen corn, a third polypeptide of 14,500 molecular weight was identified as a major in vitro product. It was preferentially labeled with methionine and slightly larger than a similar peptide in the zein fraction of corn meal. Two other polypeptides of still lower molecular weight could be detected above the background of probably incomplete polypeptides.

Journal Article↗
PubMed1977

Density differences between membrane and secreted immunoglobins of murine splenocytes.

The buoyant densities of mouse immunoglobulins were determined by isopycnic centrifugation in phosphate-buffered cesium chloride using beta-galactosidase as marker. The buoyant densities of IgG, TEPC 15 IgA, secreted IgM, and MOPC 104E IgM were consistent with their carbohydrate contents both in the presence and the absence of the nonionic detergent, Nonidet P-40. Intracellular IgM from spleen cell lysates had a buoyant density corresponding to a carbohydrate content of 6%. Membrane IgM from detergent lysates of spleen cells was less dense than either intracellular or secreted IgM in the presence of detergent. The IgD-like membrane molecules were more dense than membrane IgM.

Animals↗