Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell. Although both mother and daughter begin G 1 simultaneously, the mother cell progresses through G1 more rapidly. Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START. We present an alternative model in which the daughter cell-specific Ace2 transcription factor delays G 1 in daughter cells. Deletion of ACE2 produces daughter cells that proceed through G 1 at the same rate as mother cells, whereas a mutant Ace2 protein that is not restricted to daughter cells delays G1 equally in both mothers and daughters. The differential in G1 length between mothers and daughters requires the Cln3 G1 cyclin, and CLN3-GFP reporter expression is reduced in daughters in an ACE2-dependent manner. Specific daughter delay elements in the CLN3 promoter are required for normal daughter G 1 delay, and these elements bind to an unidentified 127-kDa protein. This DNA-binding activity is enhanced by deletion of ACE2. These results support a model in which daughter cell G 1 delay is determined not by cell size but by an intrinsic property of the daughter cell generated by asymmetric cell division.

The influenza A virus polymerase transcribes and replicates the eight virion RNA (vRNA) segments. Transcription is initiated with capped RNA primers excised from cellular pre-mRNAs by the intrinsic endonuclease of the viral polymerase. Viral RNA replication occurs in two steps: first a full-length copy of vRNA is made, termed cRNA, and then this cRNA is copied to produce vRNA. The synthesis of cRNAs and vRNAs is initiated without a primer, in contrast to the initiation of viral mRNA synthesis, and requires the viral nucleocapsid protein (NP). The mechanism of unprimed viral RNA replication is poorly understood. To elucidate this mechanism, we used purified recombinant influenza virus polymerase complexes and NP to establish an in vitro system that catalyzes the unprimed synthesis of cRNA and vRNA using 50-nucleotide-long RNA templates. The purified viral polymerase and NP are sufficient for catalyzing this RNA synthesis without a primer, suggesting that host cell factors are not required. We used this purified in vitro replication system to demonstrate that the RNA-binding activity of NP is not required for the unprimed synthesis of cRNA and vRNA. This result rules out two models that postulate that the RNA-binding activity of NP mediates the switch from capped RNA-primed transcription to unprimed viral RNA replication. Because we showed that NP lacking RNA-binding activity binds directly to the viral polymerase, it is likely that a direct interaction between NP and the viral polymerase results in a modification of the polymerase in favor of unprimed initiation.

Transcription of the CLN3 G 1 cyclin in Saccharomyces cerevisiae is positively regulated by glucose in a process that involves a set of DNA elements with the sequence AAGAAAAA (A 2 GA 5 ). To identify proteins that interact with these elements, we used a 1-hybrid approach, which yielded a nuclear zinc finger protein previously identified as Azf1. Gel shift and chromatin immunoprecipitation experiments show that Azf1 binds to the A 2 GA 5 CLN3 regulatory sequences in vitro and in vivo, thus identifying a transcriptional regulatory protein for CLN3 and a DNA sequence target for Azf1. We show that glucose-induced expression of a reporter gene driven by the A 2 GA 5 CLN3 regulatory sequences is dependent upon the presence of AZF1. Furthermore, deletion of AZF1 markedly reduces the transcriptional induction of CLN3 by glucose. In addition, Azf1 can induce reporter expression in a glucose-specific manner when artificially tethered to a promoter via the DNA-binding domain from Gal4. We conclude that AZF1 is a glucose-dependent transcription factor that interacts with the CLN3 A 2 GA 5 repeats to play a positive role in the regulation of CLN3 mRNA expression by glucose.Saccharomyces cerevisiae grows rapidly in glucose medium. With depletion of glucose, growth slows dramatically, as the cells move from glucose fermentation to oxidative metabolism of ethanol. The carbon source affects both the rate of growth in cellular mass and the rate of progression through the cell cycle. Cells rapidly increasing in mass on glucose medium proceed rapidly through the cell cycle, while cells slowly increasing in mass on nonfermentable carbon sources have a correspondingly prolonged cell cycle. This adjustment of the cell cycle length by nutrients is accomplished primarily by regulating the length of time that the yeast cells spend in G 1 phase (16,18).The G 1 cyclin encoded by CLN3 provides one of several links between growth in mass and proliferation. Changes in CLN3 expression alter G 1 duration: increased expression of CLN3 shortens G 1 , while loss of CLN3 prolongs G 1 (7,19). CLN3 expression is in turn regulated by nutrients through several distinct pathways. Both the Tor phosphatidylinositol 3-kinase and the RAS/GPA2/cyclic AMP (cAMP) pathways are thought to regulate Cln3 translation through effects on protein synthesis rates. Translation of CLN3 is especially sensitive to decreases in the abundance of translational initiation complexes (1,13,14,22). Since both of these pathways regulate pleiotropic responses to nutrients, these mechanisms can accelerate both growth in mass and proliferation in response to nutrient signals.In addition to translational regulation, nutrients affect transcription of CLN3. CLN3 mRNA levels are induced by glucose and decrease in the presence of nonfermentable carbon sources. This involves a set of repeated sequences upstream of the CLN3 coding region. Induction of CLN3 mRNA levels by glucose does not require active growth, and it is not blocked by loss of the Ras/cAMP pathway (21) or by blocking the To...

Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G 1 .