Hybridization properties of sequences adjacent to triphosphorylated 5'-ends of nuclear pre-mRNA from mouse Ehrlich carcinoma.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method /1/. These fragments as well as fragments of total pre-mRNA of the same size were polyadenylated in vitro by ATP:RNA adenyltransferase and used as templates for the synthesis of [32P] cDNA by reverse transcriptase in the presence of an oligo(dT) primer. The use of cDNA transcribed from the triphosphorylated 5'-end fragments of pre-mRNA (5'-cDNA) and from the total pre-mRNA fragments allows one to calculate the complexity of the 5'-end fraction pre-mRNA and to detect these sequences in polysomal mRNA. Sequences adjacent to 5'-phosphorylated ends of pre-mRNA represent a specific class of sequences with a complexity of about 200 kb. It was also found that about 25% of total pre-mRNA and about a half of sequences adjacent to triphosphorylated 5'-ends are present in polysomal mRNA. A high homology between triphosphorylated 5'-end fragments of pre-mRNA and mRNA sequences may be explained in terms of splicing. Less than 30% of 5'-cDNA hybridized to moderately repetitive DNA while most of them are represented by unique DNA sequences. About 15% of 5'-cDNA contained oligo(dA) sequences originated from oligo(U) in pre-mRNA from which it was transcribed.