Determination of the 5'-terminal sequence of tobacco vein mottling virus RNA and mapping of the genome-linked protein

Thesis/Dissertation · Thu Jan 01 00:00:00 EST 1987

OSTI ID:7164248

Shahabuddin, M

The nature of the 5'-terminus of the potyvirus tobacco vein mottling virus RNA (TVMV) was investigated. Recombinant plasmids containing cDNA fragments from the 5'-terminal region of TVMV RNA were constructed and used to transform Escherichia coli. Clones containing the desired plasmids were selected by hybridization. To localize the point of attachment of the genome-linked protein (VPg), /sup 125/I-labeled TVMV RNA was partially hydrolyzed and used as hybridization probe against the plasmid containing 5'-terminal sequence and four previously described plasmids containing TVMV cDNA. Together these five plasmids contained about 90% of the viral genomic RNA sequence. Hybridization was strongest to plasmids containing the more 5'-terminal sequences, suggesting that the location of the VPg was at or neat the 5'-terminus of the RNA. About 1140 nucleotides of 5'-terminal RNA sequence were determined by a combination of dideoxynucleotide chain termination, wandering spot analysis and base-specific chemical degradation methods. The location of the VPg cistron in the TVMV genome was also investigated. A partial N-terminal amino acid sequence of TVMV VPg, radiochemically modified with /sup 125/I-Bolton-Hunter reagent, was determined. Lysyl residues were found at positions 2, 11 and 16. A search within the TVMV polyprotein sequence for a similar distribution of lysyl residues revealed a unique location, which is at the putative N-terminus of the NI/sub a/ protein.

• Research Organization: Kentucky Univ., Lexington (USA)
• OSTI ID: 7164248
• Resource Relation: Other Information: Thesis (Ph. D.)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LABELLED COMPOUNDS
HYBRIDIZATION
PROTEINS
AMINO ACID SEQUENCE
RNA
STRUCTURAL CHEMICAL ANALYSIS
ESCHERICHIA COLI
GENES
IODINE 125
PLASMIDS
VIRUSES
BACTERIA
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PARASITES
RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques