ABSTRACT
A full-length cDNA clone (pMCM41) was constructed to contain the exact 5' end of MCMV behind a T7 RNA polymerase promoter and a Smal site at the 3' end. Uncapped RNA synthesized from pMCM41 has the exact 3' end of viral RNA (vRNA) but is missing the cap found on vRNA. This RNA was infectious in protoplasts from black Mexican sweet (BMS) maize (Zea mays) suspension cultures. Uncapped transcripts were also infectious when inoculated onto maize plants and produced an infection indistinguishable from vRNA-inoculated plants. Capped pMCM41 transcripts which initiated at position +2 of the cDNA clone, as well as capped or uncapped RNA synthesized from a clone containing an extra G between the T7 promoter and the 5' end of MCMV sequence (pMCM721), were less infectious than uncapped pMCM41 transcripts in BMS protoplasts. The transcripts one nucleotide longer or shorter than uncapped pMCM41 transcripts were not able to infect maize plants.
Subject(s)
DNA, Viral/genetics , Plant Viruses/genetics , RNA, Viral/genetics , Transcription, Genetic , Zea mays/microbiology , Base Sequence , Blotting, Northern , Capsid/biosynthesis , Capsid/genetics , Cloning, Molecular , Kinetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Plasmids , Polymerase Chain Reaction , Protoplasts/microbiologyABSTRACT
Maize chlorotic mottle virus (MCMV) is a 30-nm icosahedral plant virus composed of a single 25-kDa capsid protein component and a 4.4-kb single-stranded, positive-sense genomic RNA. Northern blot hybridization analysis detected a single 3'-terminal 1.1-kb subgenomic RNA in infected plants. Virion RNA directs the synthesis of several polypeptides in a rabbit reticulocyte lysate in vitro translation system of which only the 25-kDa polypeptide is immunoprecipitated by MCMV capsid protein antiserum. The 1.1-kb subgenomic RNA is a highly efficient messenger RNA for capsid protein synthesis. Positive polarity in vitro transcripts from 3'-proximal MCMV cDNA clones direct the synthesis of the capsid protein in in vitro translation experiments. These data suggest that the MCMV capsid protein is expressed from a subgenomic RNA in vivo, and that the 25-kDa capsid protein is encoded by the 3'-proximal open reading frame in the MCMV genome.
Subject(s)
Capsid/genetics , Genes, Viral , Plant Viruses/genetics , RNA, Messenger/genetics , Viral Structural Proteins/genetics , Base Sequence , Molecular Sequence Data , Oligonucleotide Probes , Open Reading Frames , Plasmids , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sequence Homology, Nucleic Acid , Zea maysABSTRACT
The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.
Subject(s)
Plant Viruses/genetics , Zea mays/microbiology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Genes, Viral , Molecular Sequence Data , RNA, Viral/genetics , Restriction Mapping , Viral Proteins/geneticsABSTRACT
The Ti plasmid DNA maintained in octopine-type crown gall tumor lines is variable, but always includes at least part of the Ti plasmid that maps over the region of Hind III fragment 1 of pTiB6-806. The right-hand boundary of transferred DNA (T-DNA) varies considerably among the three independent tumor lines examined; the left boundary was not located definitively. The T-DNA of two sibling clones of the same tumor line, E1 and E9, appears identical. The copy number of T-DNA in E9 tumor DNA appears higher for the right end (about 30 copies) than for the left end (approximately 1 copy).
