Cloning and physical mapping of DNA complementary to potato leafroll virus RNA
Potato leafroll virus (PLRV) was aphid-transmitted from potato (Solanum tuberosum cultivar Russett Burbank) to ground cherry (Physalis floridana), where it was maintained by serial aphid transmission. Serological and plant differential tests indicated that the isolate was not contaminated with beet western yellows virus. Purified PLRV RNA was poly(A)-tailed in vitro and used as a template for reverse transcriptase, primed with oligo(dT). Alkaline gel electrophoresis of /sup 32/P-labeled first-strand complementary DNA (cDNA) indicated a major size range of 0.1 to 3.5 kilobases (kb). A small percentage of transcripts corresponded to full length PLRV RNA. Following RNase H and DNA polymerase I-mediated second strand synthesis, double-stranded cDNA was cloned into the Pst I site of the plasmid pUC9 using oligo (dC)-oligo(dG) tailing methodology. Escherichia coli JM109 transformants were screened with first-strand /sup 32/P-cDNA in colony hybridization experiments to confirm that recombinants contained PLRV-specific sequences.
- Research Organization:
- Texas A and M Univ., College Station (USA)
- OSTI ID:
- 7187998
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA
HYBRIDIZATION
VIRUSES
DNA SEQUENCING
DNA-CLONING
DNA POLYMERASES
ELECTROPHORESIS
PHOSPHORUS 32
RECOMBINANT DNA
RNA
RNA-ASE
SOLANUM TUBEROSUM
TRACER TECHNIQUES
TRANSCRIPTION
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CLONING
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERASES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHODIESTERASES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PLANTS
POLYMERASES
RADIOISOTOPES
SOLANUM
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques