(8,9) or DNA recombinants (10, 11). However, the task of assigning these polypeptides to specific functions has just begun. Indeed, until now, no poxvirus proteins with known functions have been mapped by molecular or classical genetic methods.Early mRNAs of vaccinia virus that have been examined in detail do not appear to be spliced or shortened at their 5' ends (12-15). For one early mRNA, the nucleotides within the 60-bp sequence immediately upstream ofthe initiation site are 89% adenylate and thymidylate, and no homologies to prokaryotic or eukaryotic sequences were found further upstream (16). This finding led to the proposal that the vaccinia virus RNA polymerase (17, 18) has its own novel DNA recognition sequences.Although additional early vaccinia virus genes are being examined to develop a consensus sequence, such information alone will be insufficient to establish functional significance.In vitro mutagenesis provides a direct way ofcorrelating the two parameters: nucleotide sequence and function. Such studies are facilitated by the use of selectable genetic markers. For vaccinia virus, the most suitable marker is thymidine kinase (TK). Dubbs and Kit (19) demonstrated that TK activity increases after infection of TKV or TK-mouse L cells with wildtype vaccinia virus but not with a TK-mutant, providing evidence that the enzyme is virus-coded. However, no information existed regarding the genetic locus of the vaccinia TK. For herpesvirus, which also encodes TK (20)

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TKnegative nonsense mutants.

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