The nucleotide sequence of the DNA encoding the traM, finP and the promoter proximal segment of the traJ gene of the F plasmid has been determined. The predicted amino acid sequence for the traM protein shows that this inner membrane protein contains no signal sequence. The promoters for both the traM and traJ genes have been mapped by in vitro transcription and nuclease S1 protection experiments. No unambiguous location can be assigned to the finP gene but all candidates, if translated, would encode small proteins of between 24 and 52 amino acids.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT, has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT‐rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand‐specific nicks acquired by a lambda oriT genome upon propagation in Flac‐carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .

A restriction endonuclease map of EcoRI fragment f6 of F sex factor DNA was constructed and aligned with pre-existing physical and genetic maps. Results of genetic complementation tests and analysis of proteins synthesized in minicells from PstI and BglII sub-fragment clones, or from a specific BglII fragment deletion, have allowed mapping of the locations of the origin of DNA transfer and many of the transfer genes known to lie on f6. The proteins detected account for 78% of the coding capacity of fragment f6.

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The thymidine kinases encoded by herpesviruses of higher vertebrates form a distinct group and are unrelated to the thymidine kinases (TKs) of other organisms. Their evolutionary source has not been identified, but our analysis has revealed a clear relationship with a sequence of human deoxycytidine kinase (dCK) published recently. We report the sequence of the putative TK of channel catfish virus, a herpesvirus of a lower vertebrate, and show that it is also related to dCK. We propose, therefore, that the TKs of herpesviruses of higher and lower vertebrates have evolved, either independently or successively, from a cellular dCK.

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SUMMARYA homology search of proteins predicted from the recently reported complete DNA sequence of varicella-zoster virus (VZV) revealed that the product of gene 13 was highly homologous to eukaryotic and prokaryotic thymidylate synthetases (TSs). The VZV protein was shown to be a TS by three functional tests. Firstly, a plasmid designed to express the native protein was able to complement a strain of Escherichia coil in which the natural TS gene is deleted. Secondly, in an enzyme assay for TS, extracts of the complemented strain were capable of releasing tritiated water from 2'-deoxy [5-3H]uridylate. Thirdly, these extracts contained a protein that bound isotopically labelled 5-fluoro-2'-deoxyuridylate, a ligand specific for the active site of TS. In addition, a novel ligand-binding protein was detected in human cells infected with VZV.Two common human diseases, chickenpox and shingles, are caused by varicella-zoster virus (VZV). This virus is a member of the Alphaherpesvirinae, a subfamily of the Herpesviridae typified by the most extensively studied human herpesvirus, herpes simplex virus type 1 (HSV-1). Davison & Scott (1986) identified genes encoding 67 unique proteins from an analysis of the complete sequence of the 125 kbp linear double-stranded DNA genome of VZV. Five glycoprotein genes were proposed from considerations of primary amino acid sequence, and the glycoprotein products of four have been detected (Ellis et al., 1985;Davison et al., 1985;Keller et al., 1986Keller et al., , 1987. Functional assignments for 12 additional VZV proteins are dependent on comparisons with available data for HSV-1 proteins of known function (Davison & Scott, 1986). The functions of two of the remaining 50 VZV proteins may be proposed on the basis of homology with non-herpesvirus proteins. One is related to several eukaryotic protein kinases (McGeoch & Davison, 1986), and the other is homologous to prokaryotic and eukaryotic thymidylate synthetases. In this paper, we describe the structural and functional identification of the VZV thymidylate synthetase (TS) gene and the detection of a novel TS in VZV-infected cells.TS (5,10-methylenetetrahydrofolate : dUMP C-methyltransferase, EC 2.1.1.45) is a homodimeric enzyme which catalyses the reductive methylation of deoxyuridylate to thymidylate. As the sole means for supplying thymidine nucleotides de novo, TS is crucial in DNA metabolism. A particularly striking feature of this enzyme is its high degree of amino acid sequence conservation. For example, the human and Escherichia coli proteins are 53% homologous (Takeishi et al., 1985). A computer-aided homology search of the 67 VZV proteins revealed that the product of gene 13 has a similar degree of homology to published TSs. The protein-coding t Present address:

A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.