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The cryptopleurine resistance gene, cry1, of Saccharomyces cerevisiae has been molecularly cloned using genetic complementation of cryptopleurine sensitivity by the cryptopleurine resistance gene contained in a clone library prepared from DNA of a cryptopleurine resistant strain. Analysis of RNA transcripts indicated that the cry1 gene is the template for a transcript of approximately 900 bases and that the primary transcript contains an intron of approximately 300 bases. In vitro hybrid selection translation experiments indicated that this transcript encodes a protein of molecular weight 17 kilodaltons which on two-dimensional SDS polyacrylamide gels exactly coincides with ribosomal protein rp59. Further analysis showed that when the gene was present on a plasmid of about five copies per cell the amount of messenger RNA was elevated approximately five-fold compared to a cell that had only a single chromosomal copy. The rate of synthesis of ribosomal protein rp59 was not detectably elevated. These data suggest that the cry1 gene is regulated, at least in part, post-transcriptionally.

Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide. Two of these regions have been molecularly cloned. Each MATERIALS AND METHODSEscherichia coli K-12 strains JF1754 (hsdR, lac, gal, metB, leuB, hisB) and HB101 (hsdS20, recA13, proA2, lacYl, galK2, rpsL20, supE44) were used as hosts for plasmid propagation. Yeast DNA for Southern blots was purified as described (12). S. cerevisiae diploid strain JH101 (MATa/MATa, adel/ADEI , leu2-3,2-112/ LEU2', ura3-52/ura3-52, HIS4+/his4-912) was constructed for this study. pBR325 (13) In Drosophila melanogaster a series of a-amanitin-resistant, lethal, and temperature-sensitive mutations affecting RNA polymerase II activity (EC 2.7.7.6) have all been mapped to a single genetic locus, RpII (1,2). By using the DNA of the transposable element P as probe, DNA sequences from a mutant strain of D. melanogaster bearing a lethal P-element insertion in this RpII locus were cloned (3). Of the four different in vivo transcripts that originate from this RpII region of D. melanogaster DNA, only one has a homolog in mammalian DNA (4). In interspecies DNA-mediated gene transfer experiments this conserved sequence was shown to encode the gene conferring sensitivity to inhibition of RNA polymerase II activity by a-amanitin (4). These latter studies (4) clearly identified the DNA of a conserved RNA polymerase II structural gene. This gene in D. melanogaster encodes the largest (Mr = 215,000) subunit of RNA polymerase 11 (5).The conservation of eukaryotic RNA polymerase II subunit structure and antigenicity extends to fungal species such as Saccharomyces cerevisiae (6-11). We have therefore asked if yeast RNA polymerase II DNA could be detected by using the DNA of the D. melanogaster RpII region as a probe. Unexpectedly, not one but three different loci were detected in the S. cerevisiae genome when the D. melanogaster RNA polymerase II DNA was used as a probe. These yeast DNA sequences thus appear to be three members of a family of related genes. We suggest that they encode subunit polypeptides of RNA polymerases I, II, and III. RESULTS Cloning Yeast DNA Homologous to a D. melanogaster RNA Polymerase II Gene. Two subclones of D. melanogaster DNA, p4.1 and p4.2, together contain most of the structural gene information for the largest subunit of D. melanogaster RNA polymerase 11 (4, 5). We determined whether related sequences could be detected by hybridization to S. cerevisiae genomic DNA by using either of these D. melanogaster DNA species as probe. Yeast DNA (20 ,ug) was digested with the restriction endonuclease EcoRI and fractionated by electrophoresis in agarose gels; a nitrocellulose blot of this was probed under various degrees of stringency with nicktranslated p4.2, a plasmid in which the D. melanogaster DNA is carried on pBR325. A number of hybridizing fragments were seen (Fig. 1A, lanes a and b) when the hybridization so...

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45' transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.Omnipotent suppressor mutants of the yeast Saccharomyces cerevisiae are named for their ability to suppress simultaneously UAG, UGA, and UAA nonsense mutations. These suppressors were first identified by IngeVechtomov and Andrianova (19) and were mapped to two loci that have been designated sup35 and sup45 (15), sup2 and supi (19), or supP and supQ (10). sup45 and a more recently isolated omnipotent suppressor, sup46 (31), map near lys2 on chromosome 2R but are presumably distinct (31), and sup35 is on chromosome 4R (15). Unlike tRNA suppressors, omnipotent suppressors are usually recessive; they display a variety of allele-specific pleiotropic effects in vivo, including osmotic sensitivity, high or low temperature sensitivity, respiratory deficiency, and sensitivity to aminoglycoside antibiotics such as paromomycin (15,19,46,47). Ribosomes isolated from omnipotent suppressor strains have been reported to show increased misreading in vitro (43,44) and, at least for sup46 (26), this misreading is enhanced by paromomycin. Paromomycin induces phenotypic suppression of nonsense and presumed missense mutations (4, 42) and has been shown to decrease the fidelity of translation in vitro, both in S. cerevisiae (33, 42) and in Escherichia coli (5) by binding to ribosomes (33). In vivo, sup45 and paromomycin have been shown to act synergistically in their suppression of nonsense mutations of nutritional markers (46). Ribosomes purified from sup] (sup45) mutant strains show an increased dissociability into subunits in vivo (44).One low-temperature-sensitive allele of supi (reference 45) is defective in 60S subunit assembly. All in all, th...

ZEBRA has been shown to activate model reporter genes consisting of synthetic oligomerized ZEBRA response elements upstream of a minimal CYCI promoter fused to I-galactosidase in the yeast Saccharomyces cerevisiae. Here it is shown that in S. cerevisiae ZEBRA activates transcription of natural Epstein-Barr virus promoters. Two Epstein-Barr virus promoters were shown to be activated by ZEBRA in S. cerevisiae: Zp, the promoter that regulates expression of BZLF1, which encodes ZEBRA; and EAp, the promoter controlling expression of BMRF1, which encodes diffuse early antigen, EA-D. These observations indicate that neither mammalian-specific nor virally encoded coactivators are obligatory for ZEBRA to stimulate expression from these two promoters. Zp was also strongly activated by endogenous yeast factors. EAp was not activated by yeast factors. The results show that in S. cerevisiae and in B cells, ZEBRA dominates the response of EAp; ZEBRA plus endogenous cell factors activate Zp. Epstein-Barr virus (EBV) immortalizes human B lymphocytes and thereafter remains in a latent state of limited gene expression (23, 31). Inducing agents, including phorbol esters, sodium butyrate, calcium ionophores, and anti-immunoglobulin, activate the lytic EBV life cycle in B lymphocytes in vitro (9, 21, 29, 49). Induction of the lytic phase of viral growth results in an orderly cascade of expression of immediate-early and early genes, followed by lytic viral DNA replication, expression of late genes, and viral maturation (23, 31). ZEBRA , the immediate-early lytic cycle gene product, is pivotal in this cascade (6, 32). Induction of the lytic cycle is accompanied by expression of the BZLF1 gene encoding ZEBRA (45). ZEBRA then activates its own transcription as well as transcription of several viral early genes (5, 7, 10, 12, 26, 27, 36, 39, 44). ZEBRA also plays a direct role in lytic viral DNA replication by binding to the lytic replication origin, oriLyt (11, 40, 41). ZEBRA, a DNA-binding transcriptional activator, is a member of the bZIP family (4, 10, 25). ZEBRA binds to the same heptamer DNA site (TGAGTCA) that is recognized by cellular AP-1 bZIP transcription factors (4, 10, 25). ZEBRA also binds degenerate heptamer sequences called ZEBRA response elements (ZREs), whose consensus is T G/t A/T G C/T A/C/g A. Many ZREs are not bound by mammalian cellular AP-1 transcription factors, such as Fos (25, 27, 46). EBV genes that respond to ZEBRA in B cells contain ZREs and AP-1 sites in various numbers, configurations, and spacings in their 5' upstream regions (7, 22, 26, 36, 39, 44). It is not yet understood what combination, orientation, spacing, or affinity of AP-1 sites and ZREs confers ZEBRA responsiveness to a gene. Nor is it known whether ZEBRA alone activates its responsive genes, in concert with the basal transcription machinery, or whether the combinatorial action of ZEBRA and other general or specific cellular transcriptional activators is