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To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.

The structural integrity and biological activity of many proteins are known to depend on their content of one or a very few tightly bound low-molecular-weight moieties, often metal ions. There is a growing appreciation of a strong analogy between the factors that determine and affect the molecular structure of proteins and the corresponding factors for nucleic acids. Thus, despite wide differences in the chemical nature of the monomers of which they are formed, the major determinant of their native conformation is their primary structure.1-3 The bonding forces and environmental factors on which depend the integrity of their conformations also appear to be the same, and they are responsive to similar denaturants.4 The analogy is particularly striking in the case of sRNA,5 whose small size is conducive to a unique macromolecular architecture containing elements of both secondary and tertiary structure6' 7(rather than to the constellation of statistical structures likely for ribosomal, viral, and messenger RNA's). The finding reported here, that several sRNA's (when prepared by conventional methods involving denaturing steps) require the incorporation of site-bound Mg++ or some other divalent cation in order to be able to express their amino acid acceptor activities, makes the analogy seem even more plausible.While the involvement of Mg++ in the structure and function of sRNA has been

SUMMARYThe effect of interferon on expression of Epstein-Barr virus (EBV) early gene functions was investigated. The' early antigen' synthesis which follows either EBV superinfection of established lymphoid cell lines or 5'-iododeoxyuridine activation of the intrinsic EBV genomes harboured by these cells could be suppressed with interferon. In contrast, the spontaneous early antigen expression that occurs in a few per cent of the cells in the producer cell lines could not be blocked with interferon.The lymphoid cell lines tested differed in their ability to acquire an antiviral state after exposure to interferon. Several cell lines were also growth inhibited by the interferon preparations. The antiviral and growth inhibitory activities of different interferon preparations could not be separated by a number of criteria.

DNA replication of 2 human lymphoid cell lines (U296 and Raji), latently infected with the Epstein-Barr virus, has been compared using a density transfer approach. Typical of non-malignant lymphoblastoid cells, U296 cells divided once in bromodeoxyuridine-supplemented medium to form hybrid but not heavy-density host DNA. Replication of the intracellular Epstein-Barr virus DNA was selectively inhibited in these cells with only 15% of the viral genomes duplicating once to form hybrid-density viral DNA. However, some heavy-density viral DNA was formed in the U296 cells and DNA synthesis can thus initiate again on newly duplicated viral genomes in cells that have traversed only a single S phase. These results contrast strongly with observations concerning the Burkitt-lymphoma-derived cell line. Lymphoma cells are not growth-inhibited and most of the latent Epstein-Barr virus genomes of the Raji line replicated once, and only once, in successive S phases. While the majority of the 50 Epstein-Barr virus genomes of both the Raji and U296 cell lines are maintained as extra-chromosomal DNA plasmids, the control of their duplication is distinctly different in the respective malignant and non-malignant host cells.

A high-density fraction of high-molecular- After an additional 40 hr of incubation, the cells were harvested and washed twice with sodium phosphate-buffered saline. The cells were then suspended at a concentration of 107 cells per ml in phosphate-buffered saline and lysed by the addition of 0.5 volume of 3% Sarkosyl (Geigy) in 75 mM Tris HCl/25 mM EDTA (pH 9.0). After gentle rolling to obtain a clear, viscous lysate, 0.1 volume of 1% Pronase was added, and the mixture was incubated for 2 hr at 37°. The solution was then cooled to room temperature (210) and diluted with 15 volumes of deaerated 50 mM Tris HCl (pH 8.5). After 1 hr of slow swirling to obtain a homogeneous solution containing 5,/g/ml of DNA, solid CsCl was added and dissolved in the viscous DNA solution. In some experiments, the DNA solution was further treated with phenol as described (7), followed by dialysis, prior to addition of the CsCl.EBV [3H]DNA (7 X 104 cpm/ug) was isolated from virus particles recovered from the P3HR-1 cell line as described (7).[

The replication of the 50 to 60 latent, predominantly extrachromosomal, Epstein-Barr virus genomes maintained by the Burkitt-lymphoma-derived Raji cell line was investigated by using a Meselson-Stahl density transfer approach. Samples of DNA isolated from cells cultivated for different periods in bromodeoxyuridinesupplemented medium were fractionated according to density, and the distribution of viral and cellular DNAs among the heavy-, hybrid-, and light-density species was quantitated. The results indicate that the majority of latent Epstein-Barr virus DNA plasmids each replicate once during the cell cycle.

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