We showed previously that the increased rate of mRNA turnover associated with premature translational termination in the yeast Saccharomyces cerevisiae requires a functional UPF1 gene product. In this study, we show that the UPFI gene codes for a 109-kDa primary translation product whose function is not essential for growth. The protein contains a potential zinc-dependent nucleic acid-binding domain and a nucleoside triphosphate-binding domain. A 300-amino-acid segment of the UPF1 protein is 36% identical to a segment of the yeast SEN1 protein, which is required for endonucleolytic processing of intron-containing pre-tRNAs. The same region is 32% identical to a segment of Mov-10, a mouse protein of unknown function. Dominant-negative upfl mutations were isolated following in vitro mutagenesis of a plasmid containing the UPF1 gene. They mapped exclusively at conserved positions within the sequence element common to all three proteins, whereas the recessive upfl-2 mutation maps outside this region. The clustering of dominant-negative mutations suggests the presence of a functional domain in UPF1 that may be shared by all three proteins. We also identified upf mutations in three other genes designated UPF2, UPF3, and UPF4. When alleles of each gene were screened for effects on mRNA accumulation, we found that the recessive mutation upJ3-1 causes increased accumulation of mRNA containing a premature stop codon. When mRNA half-lives were measured, we found that excess mRNA accumulation was due to mRNA stabilization. On the basis of these results, we suggest that the products of at least two genes, UPF1 and UPF3, are responsible for the accelerated rate of mRNA decay associated with premature translational termination.In a wide variety of organisms, mRNAs transcribed from genes containing nonsense or frameshift mutations accumulate to a much lesser extent than do the corresponding wild-type mRNAs. In Saccharomyces cerevisiae, the introduction of a premature stop codon into a transcript causes a reduction in mRNA half-life that leads to a decrease in steady-state mRNA accumulation (29,34,48). The introduction of an efficient tRNA nonsense suppressor, which promotes read-through and restores translation of the mRNA, prevents the decline in stability and accumulation caused by premature translational termination (34). These results suggest the existence of a mechanism that serves to adjust the intrinsic rate of mRNA decay according to the ability of the mRNA to be translated. The underlying molecular basis for such a mechanism has not yet been established.To further study how mRNA turnover is related to premature translational termination, we took advantage of a selection scheme capable of yielding mutations that uncouple the two processes. The mutations were obtained in a strain containing his4-38, a + 1 frameshift near the 5' end of the HIS4 transcript that causes translational termination at an adjacent downstream stop codon (7, 13). The his4-38 mutation results in a four-to fivefold decrease in mRNA stability (29)...

Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II). This change is reversible and does not involve a simple mutation. The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis. Thus, S. cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.

Loss of function of any one of three UPF genes prevents the accelerated decay of nonsense mRNAs in Saccharomyces cerevisiae. We report the identification and DNA sequence of UPF3, which is present in one nonessential copy on chromosome VII. Upf3 contains three putative nuclear localization signal sequences, suggesting that it may be located in a different compartment than the cytoplasmic Upfl protein. Epitope-tagged Upf3 (FLAG-Upf3) does not cofractionate with polyribosomes or 80S ribosomal particles. Double disruptions of UPF1 and UPF3 affect nonsense mRNA decay in a manner indistinguishable from single disruptions. These results suggest that the Upf proteins perform related functions in a common pathway.Several genes have been identified in Saccharomyces cerevisiae and Caenorhabditis elegans that are required for the accelerated rate of decay that occurs when translation terminates prematurely because of frameshift or nonsense mutation (1-5). Nonsense mRNA decay has been observed in a wide range of eukaryotic organisms and may contribute to the etiology of disease processes in humans. A form of ,3-thalassemia common in human Mediterranean populations has been shown to result from an amber (UAG) nonsense mutation that reduces 13-globin mRNA accumulation and may exacerbate the symptoms of the disease (6). The effects of nonsense mutations that arise in somatic cells could also be exacerbated because rapid decay ensures complete loss of function of a mRNA that might otherwise produce some functional product (3).In S. cerevisiae, mutations in UPF, UPF2, and UPF3 prevent nonsense mRNA decay (1, 2, 4, 5). They were isolated as allosuppressors of his4-38, a + 1 frameshift mutation in the HIS4 gene that causes premature translational termination (7). UPF1 codes for a 109-kDa protein that contains putative RNA binding domains, suggesting the potential for direct interaction with mRNA (2). UPF1 behaves like a soluble factor that associates with polyribosomes, but is much less abundant than individual ribosomes (8). UPF2 codes for a 126-kDa protein that functions in the cytoplasm (4, 5). The UPF2 gene was identified among clones retrieved by a two-hybrid screen using UPF] DNA as bait, indicating that the Upfl and Upf2 proteins may interact physically (4).To understand how Upf3 might be related to Upfl and Upf2, we have cloned the UPF3 gene, determined the DNA sequence,t and shown that the gene product is not essential for growth. Phenotypic analyses of single and double mutants suggest that both genes may be required in the same pathway. MATERIALS AND METHODSStrains, Plasmids, Genetic Techniques, and Media. The following strains of S. cerevisiae were used: PLY100 (MATa ura3-52 trpl-7 leu2-3,-1]2), PLY107 (MA Ta his4-38 SUF-] ura3-52 leu2 trpl-Al lys]-]), PLY140 (MATa his4-38 SUF-l upf3-1 trpl-1), BSY12 (MATa his4-38 SUFl-] upf3-1 ura3-52 trpl-1), BSY202 (MATa his4-38 upf3-1 ura3-52 leu2-2 trplrpbl-1), BSY1001 (MATa trpl-Al his4-38 SUFl-] upf3-A] ura3-52 lysl-l leu2), BSY1044 (MATa ura3-52 trpl-7 leu2-3,-112 upf3-...

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, the 25 S, 18 S, and 5.8 S ribosomal RNAs (rRNAs) derive from the post-transcriptional cleavage of a large prerRNA precursor, the 35

The expressed sequence tags (ESTs) referenced in this report are the first transcriptomes in a leaf from a half-shade ginseng plant. A cDNA library was constructed from samples of the leaves of 4-year-old Panax ginseng plants, which were cultured in a field. The 2,896 P. ginseng cDNA clones represent 1,576 unique sequences, consisting of 1,167 singletons and 409 contig sequences. BLAST comparisons of the cDNAs in GenBank's non-redundant databases revealed that 2,579 of the 2,896 cDNAs (89.1%) exhibited a high degree of sequence homology to genes from other organisms. The majority of the identified transcripts were found to be genes related with energy, metabolism, subcellular localization, and protein synthesis and transport. The chlorophyll a/b-binding protein ESTs in the ginseng leaf samples manifested a substantially higher level of expression than was observed in other plant leaves. The ESTs involved in ginsenoside biosynthesis were also identified and discussed.

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.The fission yeast Schizosaccharomyces pombe is a haploid eukaryotic microorganism whose cells exhibit two different mating types, called P (plus) and M (minus) (18,33). The mating type is determined by the genetic information in the mat1 locus, which contains either the mat1M or the mat1P allele, each of which encodes two transcripts essential for controlling cell type (24). A copy of the mating type genetic information is also present at the mat2P and mat3M"donor" loci that are located centromere-distal to mat1 and are transcriptionally silent. The mating type switches when a copy of either the mat2P or mat3M DNA transposes and substitutes for mat1 by DNA recombination, where the transposed genetic information is expressed (6,12,26).Switching occurs spontaneously at a high frequency in "homothallic" strains (h 90 , i.e., homothallic, 90% sporulation) during mitotic growth, and it follows an interesting nonrandom pattern within a cell lineage (17,28,34). Division of an unswitchable parental cell produces two daughter cells, one of which is programmed to be switching competent and the other of which is not. After cell division of a switching-competent cell, a switched and a switching-competent daughter cell are generated. On the other hand, a switching-noncompetent cell undergoes a cell division to produce a switching-competent and a noncompetent cell. Therefore, this switching pattern in the cell lineage results in only one cell switching among four granddaughter cells after two consecutive asymmetrical cell divisions of an unswitchable cell. These rules of so-called onein-four and consecutive switching a...