Analysis of expression of adenovirus DNA (fragments) by microinjection in Xenopus oocytes. Independent synthesis of minor early region 2 proteins.
Injection of whole adenovirus DNA into Xenopus oocytes results in the synthesis of large amounts of the early region 2A DNA-binding protein (E2A-DBP) and smaller amounts of polypeptide IX. The lack of synthesis of any functional messenger RNAs transcribed from the major late promotor at 16.3 map units is remarkable. Cleavage of the adenovirus DNA outside the E2A gene proper by restriction enzymes decreases synthesis of the DBP to about 10% of the amount produced after injection of intact DNA. On the other hand, presence of the terminal (Bellett) protein on the injected template enhances DBP synthesis considerably. Experiments with injected DNA restriction fragments, as well as reconstructed genes cloned into pBR322, indicate that efficient synthesis of DBP in oocytes requires the presence of either or both of the two main promoters from which the E2A gene is transcribed plus an intact 3' end of the gene. In the absence of any known promotor, 100-fold lower amounts of otherwise normal DBP are produced. Unlike in a regular infection, synthesis of DBP in oocytes does not require the product of the E1A gene. The same series of experiments also demonstrates that the DBP, a phosphoprotein, is the substrate of a cellular rather than a virus-encoded protein kinase. Two minor E2A proteins, although colinear with the major DBP, are synthesized independently. Synthesis of a 44,000 Mr protein, probably corresponding to the carboxy-terminal 360 amino acid residues of the DBP, is not decreased after injection of "promotorless" E2A genes. Unlike the 44,000 Mr protein, production of a 67,000 Mr protein (carboxy-terminal 483 amino acid residues) by one DNA-construct is probably directed by a T-A-T-A-A-A-T-A sequence in the vector DNA.