A modified procedure was developed which allows
RNA--
DNA hybridization reactions to be performed without the loss in translational capacity of
mRNA which accompanies hybridization at elevated temperatures or in the presence of the denaturing agent
formamide. Separated l and r strands of bacteriophage T2
DNA were hybridized in the presence of 4 M
sodium perchlorate at 37 degrees C with total
RNA from infected cells. After passage of the hybridization mixture through a
nitrocellulose column to remove single-strand
DNA and
DNA--
RNA hybrids, the eluent was measured for its capacity to promote
deoxynucleotide kinase (gene 1) synthesis in an in vitro
protein-synthesizing system derived from uninfected Escherichia coli. With this procedure, which should be of general use for any message whose product can be measured either enzymatically, immunologically, or by location in an acrylamide gel, it was demonstrated that
deoxynucleotide kinase mRNA is transcribed from the l strand of bacteriophage T2
DNA. By titrating with l strand
DNA, the number of
deoxynucleotide kinase transcripts present 9 min after T2 phage
infection at 30 degrees C was estimated to be about 38 copies per cell.