Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing

Abstract

The sequences at the splice junctions of many early region 4 (E4) mRNAs from adenovirus 2 (Ad2) were determined by analysis of cDNA clones. The cDNAs were synthesized from poly(A)+ mRNA isolated from HeLa cells early during Ad2 infection. A standard library was constructed, in pBR322, from double stranded cDNAs initiated by oligo-dT priming. Approximately 1% of total recombinants contained E4 sequences, however among eighty clones analyzed in detail, only four contained the 5' leader sequence. A second library was prepared using a new method that led to a greatly increased representation of desired clones. This method employed oligo-dT to prime the synthesis of the first strand and an oligonucleotide ligated to pBR322, whose sequence was present in the 5' leader, to prime the synthesis of the second strand. With this method the percentage of recombinants containing E4 sequences ranged between 15 and 50% of the total colonies. Virtually all of these E4 cDNA clones contained the 5' leader sequence and several hundred were analyzed by comparing the results from single channel dideoxy sequencing reactions. Nine unique sequence patterns were identified and representative clones were completely sequenced.

Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs.

“…These encode a minimum of seven E4 proteins (Fig. 3A) (7,35,37). Of these, E4-ORF3 and E4-ORF6 appear to be the key proteins since expression of one is sufficient to establish an essentially wild-type virus infection (4,11,12,14).…”

Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.

“…cDNAs spanning the introns of the 12S and 13S RNAs were isolated from a cDNA library constructed from RNA extracted from Ad2-infected HeLa cells early in infection (15). In each case, the integrity of the splice junctions (41) and protein-coding sequences was confirmed by DNA sequence determination by using the chain termination method (data not shown).…”

Cancer-selective therapy of the future: Apoptin and its mechanism of action

“…[71][72][73] E4orf4 is a small protein of 114 amino acids with no significant homology to any other known proteins. The protein has two distinct regions of amino acids in its structure: the proline rich region at the N-terminus (aa.…”