The map positions of the 5′ ends of eight late mRNAs from the vaccinia virus HindIII D fragment were determined by a combination of S1 nuclease-protection studies and by primer extension analysis. For genes D2, D8, D10, and Di 1, a single set of 5′ ends can be observed by S1 nuclease analysis which maps just upstream from the translation start site for each gene. For genes D3, D6, and D11a the situation is more complex. In addition to the ATG proximal family of protected DNA fragments, multiple larger protected DNA fragments map to sites up to several hundred base pairs upstream from the coding region. When primer extension mapping is carried out, large extended products are observed in all cases but that of gene D10. For genes D2, D8, and D13, these large DNA products are heterogenous in length and much longer than the S1 nuclease-protected DNA fragments. This has been observed previously for the late mRNA from the 11 K gene by Bertholet et al. [(1987). Cell 50, 153]. In the case of mRNA from genes D3 and D6, however, the lengths of the extended DNA primers agree with the lengths of the S1 nuclease-protected DNA fragments. Therefore, for genes D3 and D6, the 5' regions of the mRNA must be derived from transcription of the DNA sequences upstream from the coding region of each gene. Since the structures at the 5' ends of the late mRNA assume more than one form, there maybe multiple pathways for generating late mRNA.
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