DNA amplification-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites.
DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.
Mackow
E R
ER
Department of Medicine, Stanford University, CA 94305.
Yamanaka
M Y
MY
Dang
M N
MN
Greenberg
H B
HB
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D004279:DNA, Viral / Q000235:genetics*; D005784:Gene Amplification; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012401:Rotavirus / Q000235:genetics*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D004279:DNA, Viral; D015345:Oligonucleotide Probes; C060339:VP4 protein, Rotavirus