Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70 S RNA: Evidence for a Haploid Genome

Abstract

The sequence complexity of the 60-70 S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70 S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C r t ½ , was compared with the C r t ½ values for annealing of poliovirus 35 S RNA (2.6 × 10 6 molecular weight) with poliovirus-specific DNA and Sindbis virus 42 S RNA (4.3 × 10 6 molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 × 10 6 daltons for the cloned M-MuLV 60-70 S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35 S RNA subunits of M-MuLV 60-70 S RNA has a different base sequence.