Including Projects Cooperative with Other Labs
TVCV | Movement Proteins | Phytopathogenic bacteria | CaMV Recombination | CaMV Genes & Pathogenesis | Plectroviruses | Badnaviruses | Zeins | Membrane Immunoglobulins | Protein Synthesis and Secretion
Like viruses, research in this lab has evolved and continues to evolve. During his undergraduate days at the University of Chicago, Ulrich Melcher got an interest in the molecular workings of plants from the late Professor Larry Bogorad. For graduate thesis research, Melcher wanted to examine the initiation of protein synthesis in plants, but wound up examining protein secretion. Interest in protein traffic through cells, led to postdoctoral work on the biosynthesis and structure of membrane immunoglobulins, work continued in the Melcher laboratory at OSU. A return to plant work began with studies on cereal storage proteins, the zeins. Genetic engineering of cereals to improve nutritional quality would require vectors for introducing genes into plants. This realization led to exploration of the double-stranded DNA containing virus, cauliflower mosaic virus (CaMV). A complication of the use of this virus as a vector was the ready tendency for recombination. During studies of recombination of CaMV DNA in turnip, the lab stumbled on another virus, turnip vein-clearing virus, whose genome they proceeded to sequence. The inability of the related tobacco mosaic virus to spread in turnips led to an interest in movement protein structure and function. Facility in sequence analysis and comparison led to cooperation with laboratories studying mastreviruses, badnaviruses and plectroviruses. Plectroviruses are hosted by cell-wall-less bacteria that cause plant disease. The lab participated in studies of the Fletcher laboratory aimed at deciphering how the bacterium is transmitted to plants by leafhopper insects. The lab has also participated in a team effort on cucurbit yellow vine disease.
In Virus Evolution work with single viruses, the lab
- contributed evidence and analysis methods towards elucidating how overlapping genes arose (Lartey et al., 1996);
- contributed evidence supporting the idea that recombination is more successful when it occurs between coding regions than within coding regions;
- provided evidence of viral sequence selection within plant parts (Hackett et al., 2009);
- provided an early example of codivergence of viral species with hosts with which they were associated (Lartey et al., 1996);
- argued that even rapidly evolving viruses can, when examined in a broader scale, show evidence of codivergence with hosts (Wu et al., 2008);
- provided a description of the 30K superfamily of movement proteins that allowed successful prediction of movement function for viral proteins of diverse taxa (Melcher, 1990, 2000);
- provided the first estimate of the mutation rate for a pararetrovirus (Pennington and Melcher, 1993);
- is a prime mover in exploring the diversity of viruses in wild plants (Wren et al., 2006).
In Virus Evolution work with multiple viruses, the lab
- was the first to demonstrate experimentally recombination between plant viruses (Choe et al., 1985);
- demonstrated interference and selective allele loss after infection with marked isolates ( Melcher et al., 1985);
- pioneered application of methods to detect recombination between viral sequences (Chenault & Melcher, 1993);
- provided early phylogenetic evidence of recombination in RNA viruses (Lartey et al., 1996);
- demonstrated molecularly competition between isolates leading to relative fitness values (Zhang & Melcher, 1989);
- demonstrated cross protection between pararetroviral isolates (Zhang & Melcher, 1989);
- provided the first evidence of synergistic interaction between an RNA virus and a pararetrovirus (Hii et al, 2002);
In Plant Virus Molecular Biology and Genetics, the lab
- made the first identification of a plant virus gene required for transmission by an arthropod vector (Armour et al., 1983);
- provided early examples of the application of reverse genetics to plant virology (Melcher et al., 1986);
- provided early evidence of susceptibility of pararetroviral RNA to RNA splicing (Pennington & Melcher, 1993);
- devised leaf skeleton hybridization (Melcher, 1981), a method of locating anatmoically viral nucleic acid in plants, a method that led to the widley used method of tissue printing.
Bioinformatic contributions included
- a space-efficient and visually pleasing way of representing nucleotide sequences
- deomonstration of the utility of alternating multiple sequence alignment and phylogenetic tree construction in discerning relationships among distantly related proteins (movement proteins) (Melcher, 2000);
- establishment of gap translation as a way to optimally place insertions/deletions in a multiple sequence alignment (Melcher, 1990).
In Immunology, Melcher made contributions (both as part of the Uhr lab and independently) in
- discovery of the existence of murine IgD, facilitating the investigation of the role of this Ig class in immune responses (Melcher et al., 1974);
- the demonstration that membrane immunoglobulins were integral membrane proteins (Melcher et al., 1975);
- the demonstration, using isopycnic centrifugation, that membrane heavy chains were longer in their polypeptide chains than secreted heavy chains (Dobson & Melcher, 1979).
A more narrative explanation of some of these areas follows:
An unusual strain of the cosmopolitan bacterium Serratia marcescens causes cucurbit yellow vine disease (CYVD) and is carried to plants by the squash bug Anasa tristis. S. marcescens is a cosmopolitan bacterium inhabiitng a large variety of niches. Genetic and physiological factors needed by CYVD strains to cause disease in squash and to be transmitted by squash bugs were investigated.
How a phytopathogen is transmitted by insects was also the subject for cooperative investigation, with Jacqueline Fletcher and Astri Wayadande, of the transmission of Spiroplasma citri to plants by leafhoppers.
The nucleotide sequence of SVTS2, a SpV1-like virus originally isolated from S. melliferum, was analyzed in cooperation with Jacqueline Fletcher and Robert Davis. Comparison of its sequence with those of two SpV1 viruses isolated from S. citri revealed open reading frames likely essential for viral infection and, within each frame, encoded residues that are probably particularly important. SVTS2 DNA is considerably smaller than that of the other two and homology exists for only about one-half of the genome. Parts of SVTS2 DNA integrate into the host genome and the resulting mollicutes resist further infection by SVTS2.
Movement genes and proteins
- The amino acid sequences of movement proteins are highly divergent. At present, we recognize, by sequence similarities, three families of movement proteins: the tymovirus-type proteins, the triple gene block proteins, and proteins of the 30K superfamily. This classification is not all-inclusive, as some movement proteins appear not to fit in these three classes. Within the 30K superfamily, amino acid sequences are highly divergent. We explored the alignment of these amino acid sequences. Each of our predictions of movement function for proteins encoed by diverse viral families have since been vindicated. The alignment will allow the integration of results of site-specific mutation studies using a diversity of viruses. Alignment also provided clues to the three-dimensional structure of these proteins.
Rice tungro bacilliform virus (in collaboration with Ossmat Azzam and colleagues)
- Our experience in studying sequences led to an invitation to cooperate on the examination of a set of six complete nucleotide sequences of the badnavirus, rice tungro bacilliform virus (RTBV), all derived from a single population. The examination revealed that in the evolution of the population there had been numerous recombinational exchanges. We also deduced that the patterns of nucleotide substitution had changed over the time in which these isolates evolved from a common ancestor.
Turnip vein-clearing virus (TVCV)
- TVCV, a tobamovirus we discovered in turnips, was, by preliminary results, clearly different from other tobamoviruses that had had the nucleotide sequences of their genomes determined. We thus determined the complete sequence of nucleotides in its cDNA. This sequence and others of related viruses, determined independently, revealed a unique organization of genes that we feel requires the designation of a third subgroup of tobamoviruses. Phylogenetic analysis of tobamoviruses including subgroup 3 viruses revealed that at least one tobamovirus is a natural recombinant. We also created a plasmid containing TVCV cDNA from which RNA transcripts infectious to plants could be made.
- We found TVCV in turnip plants that we were using to investigate recombination in the plant pararetrovirus cauliflower mosaic virus (CaMV). CaMV virions contain an 8 kbp double-stranded circular DNA as genetic material. In plant nuclei, the DNA is in a minichromosome whose transcription produces a 35S RNA. This RNA is the template for reverse transcription, producing further CaMV DNA. The 35S RNA and a subgenomic 19S RNA are templates for translation of six or more polypeptides.
CaMV DNA, cloned in bacterial plasmid DNA vectors, is infectious to turnips when separated from the vector by restriction enzyme digestion. Pairs of site-specifically mutated CaMV DNAs, each by itself non-infectious, produce infections of turnip plants. The infections result not from complementation, but from recombination. Using multiply marked mutated CaMV DNAs, we determined that most recombination events happened during reverse transcription. Recombination also occurred when mutant and wild-type DNAs were coinoculated and when CaMV DNAs bearing inserts started infection.
Recombination probably occurs naturally, as well. We determined the nucleotide sequences of the DNAs of three isolates of CaMV. These and others available from work of others served as input for phylogenetic analysis of individual CaMV open reading frames. That the deduced phylogenetic relationships depended on which open reading frame was being analyzed suggested that recombination played a role in their evolution. Patterns of nucleotide substitution were anomalous.
CaMV Genes and Pathogenesis
- The CaMV DNA mutants used for the recombination studies were originally made to explore the functions of the open reading frames. All but one open reading frame were essential for infectivity of the DNA in the laboratory. The exception, open reading frame II, was required for transmission of the virus from plant to plant by aphids. Modifications to another did not interfere with infectivity, but did reduce the virus' competitive ability.
- In virions, CaMV DNA is closely associated with the larger of several versions of the coat protein. Assembly studies suggested that the coat protein binds single-stranded nucleic acid better than double stranded DNA. The observation is consistent with previrion particles that contain RNA that is reverse transcribed in the particle.
Storage Protein Genes
- CaMV DNA was once considered as a possible vector for introducing DNA into plants to improve the plants genetically. Among the traits targeted for improvement was the quality of seed storage proteins. We participated in studies of the zeins of maize, characterizing the genetic heterogeneity of their mRNAs, identifying a methionine-rich zein, and developing a new fractionation method.
Membrane Immunoglobulins (partly in laboratory of Jonathan Uhr)
- Zeins are synthesized on endoplasmic reticulum-bound ribosomes and sequestered in membrane-bounded protein bodies. A similar intracellular pathway is taken by immunoglobulins, except that these are externalized as secreted or membrane immunoglobulins. We established that the murine membrane IgM monomer was an integral membrane protein, that its disulfide bonding pattern was opposite of monomers derived from IgM pentamers by reduction, that the density of its heavy chain combined with its electrophoretic mobility in SDS-PAGE suggested that it had additional peptide sequence relative to secreted heavy chains. We also discovered a non-IgM immunoglobulin on the surface of murine spleen cells and characterized it as the murine equivalent of human IgD.
Protein Synthesis and Secretion (in laboratory of Joe Varner)
- The barley aleurone layer represents another tissue in which protein traffic, in this case the secretion of newly synthesized hydrolases, can be examined. Interest in obtaining a pure population of newly synthesized proteins was complicated by the release of previously synthesized proteins from the layers. N-termini of the protein populations were analyzed in a misguided attempt to determine the initial amino acid polymerized during translation. The same question, what is the initiating amino acid in eukaryotic protein synthesis, was attacked by studying the products that yeast cells generate in the presence of high concentrations of puromycin. These products turned out not to result from translation, but from metabolism of puromycin.
Last Updated: 27 January, 2010
E-mail inquiries to U. Melcher