Nucleic Acids Research, 1981, Vol. 9, No. 7 1675-1690
© 1981
MOLECULAR BIOLOGY |
Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA
Institute for Molecular Virology, St. Louis University School of Medicine 3681 Park Avenue, St. Louis, MO 63110, USA
Received November 24, 1980.
The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7Gppp removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [
-32P]ATP using poly-nucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligo-nucleotides were resolved by two dimensional paper electrophoresis-homo-chromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogeneous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.