A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-f luoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 ؋ 10 ؊5 to 4 ؋ 10 ؊5 per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500-to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientationdependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

CDC7 and DBF4 encode the essential Cdc7-Dbf4 protein kinase required for DNA replication in eukaryotes from yeast to human. Cdc7-Dbf4 is also required for DNA damage-induced mutagenesis, one of several postreplicational DNA damage tolerance mechanisms mediated by the RAD6 epistasis group. Several genes have been determined to function in separate branches within this group, including RAD5, REV3/ REV7 (Pol ), RAD30 (Pol ), and POL30 (PCNA). An extensive genetic analysis of the interactions between CDC7 and REV3, RAD30, RAD5, or POL30 in response to DNA damage was done to determine its role in the RAD6 pathway. CDC7, RAD5, POL30, and RAD30 were found to constitute four separate branches of the RAD6 epistasis group in response to UV and MMS exposure. CDC7 is also shown to function separately from REV3 in response to MMS. However, they belong in the same pathway in response to UV. We propose that the Cdc7-Dbf4 kinase associates with components of the translesion synthesis pathway and that this interaction is dependent upon the type of DNA damage. Finally, activation of the DNA damage checkpoint and the resulting cell cycle delay is intact in cdc7⌬ mcm5-bob1 cells, suggesting a direct role for CDC7 in DNA repair/damage tolerance.

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG) 50 or (CTG) 50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG) 50 or (CTG) 50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 ؋ 10 ؊4 to 18 ؋ 10 ؊4 per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G) 50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast.

Members of the mitogen-activated protein (MAP)1 kinase family including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAP kinases mediate cell growth, adaptation, and differentiation, in part, by phosphorylating and activating transcription factors that regulate genes required for execution of these programs (1). The PC12 pheochromocytoma cell line (2) provides a useful cell culture system in which to explore the mechanism by which MAP kinases control cell differentiation. Incubation of PC12 cells with nerve growth factor (NGF) leads to growth arrest and cellular hypertrophy as well as the acquisition of a neural morphology typified by extension of neurites. A molecular correlate of the morphologic differentiation of PC12 cells is induction of neurofilaments (3) that are composed of heavy, medium, and light polypeptide chains (4). Increased expression and phosphorylation (3, 5) of neurofilament proteins is thought to contribute to the extension and stabilization of neuronal processes. Moreover, the rat NFLC promoter has been characterized and shown to be induced by NGF in PC12 cells (6), indicating that transcriptional induction contributes to the increased content of neurofilament proteins. Mutational analysis of the NGF receptor has defined the requirement for collaborative signaling through phospholipase C␥ (PLC␥) and the monomeric G protein, Ras, for morphologic differentiation of PC12 cells (7,8). Similar mutational studies with ectopically expressed ␤PDGF receptors demonstrated the requirement for Ras signaling in combination with signaling through the Src family tyrosine kinases or regulation of PLC␥ (9, 10). A clearly defined action of Ras within cells is the stimulation of MAP kinase cascades including the ERK pathway (11). The ability of constitutive active forms of proximal regulators of the ERK pathway to induce morphological differentiation suggests that the ERK pathway is necessary and perhaps sufficient for morphologic differentiation of PC12 cells (12). However, mutant ␤PDGF receptors that can activate the ERK pathway, but not Src or PLC␥, failed to induce morphologic PC12 cell differentiation (10), indicating that the ERK pathway is not sufficient for growth factor-induced PC12 cell differentiation and requires collaboration with other signals. Expression of GTPase-deficient, constitutive active forms of G␣ q proteins or incubation with bone morphogenetic protein-2 induced PC12 cell differentiation accompanied by persistent activation of the JNK family of MAP kinases (13,14). Significantly, the ERKs were not activated in these settings, indicating that PC12 cell differentiation can sometimes occur independently of the ERK pathway and that the JNK pathway may

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [ 3 H]inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho[ 3 H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporterindependent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [ C]glycerophospho[ H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.The extracellular production of glycerophosphoinositol (GroPIns) represents a major pathway of phosphatidylinositol (PI) metabolism in growing cultures of Saccharomyces cerevisiae (1, 2). GroPIns accumulates in the growth medium at levels equivalent to about 25% of the amount of cellular PI, in contrast to much lower extracellular levels of the deacylated forms of the major yeast phospholipids phosphatidylcholine and phosphatidylethanolamine (1). In pulse-chase turnover experiments, Angus and Lester (1) have shown that GroPIns accounts for approximately 50% of the phosphorus and inositol lost from PI during growth. Furthermore, the release of GroPIns is regulated by glucose in the medium (2). Thus, the extracellular accumulation of GroPIns was shown to be specific, regulated, and a major route of PI turnover in a growing yeast culture. Recently, Hawkins et al. (9) confirmed that the addition of glucose to stationary-phase cultures results in the extracellular production of not only GroPIns but also low levels of GroPIns 4-phosphate and GroPIns 4,5-bisphosphate.GroPIns, GroPIns 4-phosphate, and GroPIns 4,5-bisphosphate are produced from PI, PI 4-phosphate, and PI 4,5-bisphosphate, respectively, by a phospholipase A and a lysophospholipase acting sequentially or by a phospholipas...