Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G0 state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

We have analyzed the functions of several pre-mRNA processing (PRP) proteins in yeast spliceosome formation. Here, we show that PRP5 (a DEAD box helicase-like protein}, PRP9, and PRPll are each required for the U2 snRNP to bind to the pre-spliceosome during spliceosome assembly in vitro. Genetic analyses of their functions suggest that they and another protein, PRP21, act concertedly and/or interact physically with each other and with the stem-loop IIa of U2 snRNA to bind U2 snRNP to the pre-mRNA. Biochemical complementation experiments also indicate that the PRP9 and PRPll proteins interact. The PRP9 and PRPll proteins may be functioning similarly in yeast and mammalian cells. The requirement for ATP and the helicase-like PRP5 protein suggests that these factors might promote a conformational change {involving either the U1 or U2 snRNP) that is required for the association of U2 snRNP with the pre-mRNA.[Key Words: Splicing; pre-mRNA; spliceosome; U2 snRNP; PRP proteins] Received June 28, 1993; revised version accepted August 9, 1993.A key to understanding the mechanism and regulation of nuclear precursor messenger RNA (pre-mRNA) splicing lies in discovering the functions of numerous trans-acting factors. These factors can be grouped into two classes: the small nuclear ribonucleoprotein particles (snRNPs)--U1, U2, U4/U6, and U5--and a multitude of non-snRNP factors (for review, see Green 1991;Guthrie 1991;Ruby and Abelson 1991;Brown et al. 1992;Rymond and Rosbash 1992;Moore et al. 1993). The snRNPs and some non-snRNP factors assemble on the premRNA to form the spliceosome on which splicing occurs. U1 snRNP binds first to the pre-mRNA, followed by the U2 snRNP and, finally, by the tri-snRNP U4/US/ U6 particle. Some non-snRNP factors may become integral components of the spliceosome, whereas others may only loosely or transiently associate with the snRNPs and/or the spliceosome. The functions of these non-sn-RNP factors are particularly intriguing as their elucidation may lead to our understanding of why the premRNA splicing apparatus is so complex and requires ATP.Pre-mRNA splicing occurs in two transesterification reactions, which are mechanistically the same as those of the group II self-splicing introns (for discussion, see 4Corresponding author.Weiner 1993). As the group II selfsplicing introns require no nucleotide or protein cofactors in vitro (for review, see ]acquier 19901, it is thought that pre-mRNA splicing is likely to be catalyzed by RNA as well. The spliceosomal small nuclear RNAs (snRNAs) may have this function. Thus, it is a quandary as to the functions of the numerous snRNP and non-snRNP proteins in premRNA splicing. Some non-snRNP factors such as the mammalian ASF/SF2 (Ge et al. 1991;Krainer et al. 1991) and SC35 (Fu and Maniatis 1992), and the Drosophila transformer (Tra}, Tra2 (Tian and Maniatis 1992), and sex-lethal (Sxl) [Baker 1989) proteins function in the recognition and selection of introns and splice sites. One non-snRNP protein has been proposed to regulate the fidelity of splicing [Bu...

When exposed to DNA-damaging agents, the yeast Saccharomyces cerevisiae induces the expression of at least six specific genes. We have previously identified one damage inducible (DIN) gene as a gene fusion (din-lacZ fusion) whose expression increases in response to DNA-damaging treatments. We describe here the identification of five additional DIN genes as din-lacZ fusions and the responses of all six DIN genes to DNA-damaging agents. Northern blot analyses of the transcripts of two of the DIN genes show that their levels increase after exposure to DNA-damaging agents. Five of the din-lacZ fusions are induced in S. cerevisiae cells exposed to UV light, gamma rays, methotrexate, or alkylating agents. One of the din-lacZ fusions is induced by either UV or methotrexate but not by the other agents. This finding suggests that there are sets of DIN genes that are regulated differently.Procaryotic and eucaryotic cells respond to the stresses caused by DNA damage or inhibited DNA replication in numerous ways. In Escherichia coli three inducible systems have been characterized: the SOS response, which is quickly activated after exposure to many DNA-damaging agents and replication inhibitors (53); the adaptive response to alkylation, which is elicited by certain alkylating agents (6, 43); and the adaptive response to oxidative damage such as that caused by hydrogen peroxide (12).The SOS response involves a number of diverse functions including increased recombination and DNA repair, enhanced mutagenesis, recA protein synthesis, prophage induction, Weigle reactivation of irradiated phage, colicin production, and filamentation. At least 17 genes, some of which have been identified with specific functions, are induced during this response. These genes are coordinately regulated by the recA and lexA gene products (28,52). The two adaptive responses result in decreased lethality caused by particular types of DNA damage. They appear to operate independently of the SOS response as they are neither under the control of the lexA and recA genes nor induced by many SOS-inducing agents. The adaptive response to alkylating agents also leads to decreases in mutagenesis by such agents as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) (27). Although these three responses seem to be distinct, there may be some interaction among them as evidenced by the fact that at least two SOS genes are induced by MNNG (2, 39).Analogous inducible responses to DNA-damaging agents may be present in both lower and higher eucaryotes. The existence of an inducible DNA repair system in Neurospora crassa is suggested by the finding that cells exposed to sublethal doses of UV, X ray, or nitrous acid have an enhanced ability to rescue lethally irradiated cells when fused into a heterokaryon after treatment (49 (24) and S. cerevisiae (5) the inhibition of protein synthesis by cycloheximide immediately after gamma irradiation blocks the repair of double-strand breaks and decreases cell survival. An inducible pathway of recombination w...

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