The ROXI gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Roxl repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Roxl itselfwere found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANBIrepression. An ANBI-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the limiting, heme synthesis decreases, Roxl levels are reduced, and the hypoxic genes are derepressed. The evidence that Roxl acts directly as a transcriptional repressor is indirect. Deletion of the ROX1 gene results in constitutive expression of the hypoxic genes, as would be expected for a gene encoding such a repressor (23). Also, insertion of the Roxldependent operator site from ANBl into the GALl upstream region confers Roxl-dependent repression on the GALl gene (20). Finally, the heme repression ofANB1 requires the general repression factor Tupl (50). To determine whether Roxl acts at the level of the DNA, we examined the ability of Roxl protein expressed in and partially purified from Escherichia coli cells to bind the hypoxic DNA consensus sequence specifically. We also report the DNA sequence of the ROXI gene, which encodes a protein with the high mobility group (HMG)-like DNA-binding motif at its amino terminus. In addition, we have found that Roxl repression of ANBI requires Ssn6 as well as Tupl. MATERIALS AND METHODSStrains. E. coli JM109 and HB101 were used for plasmid constructions (24). PR745 (New England Biolabs), containing a malE deletion and a mutation in the lon protease, was used for expression of Roxl. Bacterial cells were grown on Luria broth supplemented with 100 ,ug of ampicillin per ml when appropriate (24). (MATa spoll ura3 ade6 arg4 aro7 aspS metl4 lys2 petl7 trpl, from the Yeast Stock Center) was mated to (MATa trpl-289 leu2-3 112 ura3-52 adel-100). The diploids were sporulated, and a haploid A4Tct leu2 aro7 segregant was mated to BWG1-7aAroxl (AM4Ta adel-100 his4-519

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cermzsiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G 1 , with ␣-factor, or in G 2 /M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites in SWI ؉ and swi1⌬ yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.