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The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385Bacteriol. 172: -1391Bacteriol. 172: , 1990 and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed.

The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.

In the yeast Saccharomyces cerevisiae three G1-S cyclins, or CLNs, have been identified that trigger the GI-S transition of the cell cycle. The regulation of the G~-S transition is particularly intriguing in Drosophila both because G~ is added to the cell cycle developmentally and G1-S regulators may drive the polytene cell cycle.To identify potential G~-S regulators from Drosophila, a cDNA expression library was constructed in which Kc cell cDNAs were placed in a high-copy S. cerevisiae vector under the control of the constitutive ADH1 promoter. Following transformation into an S. cerevisiae strain lacking all three CLN gene products, we identified one Drosophila cDNA that complemented the yeast G1 cyclins and restored growth to near wild-type levels. The CLNDm gene is present as a single copy in the Drosophila genome and encodes a 1.2-kb mRNA. DNA sequence analysis reveals that although this gene has cyclin homology, it is a new member of the cyclin gene family. CLNDm mRNA expression correlates with periods of maximal cell division throughout Drosophila development. The transcript is most abundant in early embryos, and it is present in low levels in larvae, pupae, and adults. Drosophila embryos hybridized in situ to this cyclin gene show uniform expression of the message throughout the embryo, with diminishing expression as embryogenesis proceeds.

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