The three budding yeast CLN genes appear to be functionally redundant for cell cycle Start: any single CLN gene is sufficient to promote Start, while the cln1 cln2 cln3 triple mutant is Start defective and inviable. Both quantitative and apparently qualitative differences between CLN genes have been reported, but available data do not in general allow distinction between qualitative functional differences as opposed to simply quantitative differences in expression or function. To determine if there are intrinsic qualitative differences between Cln proteins, we compared CLN2, CLN3, and crippled (but still partially active) CLN2 genes in a range of assays that differentiate genetically between CLN2 and CLN3. The results suggest that different potencies of Cln2, Cln3, and Cln2 mutants in functional assays cannot be accounted for by a simple quantitative model for their action, since Cln3 is at least as active as Cln2 and much more active than the Cln2 mutants in driving Swi4/Swi6 cell cycle box (SCB)-regulated transcription and cell cycle initiation in cln1 cln2 cln3 bck2 strains, but Cln3 has little or no activity in other assays in which Cln2 and the Cln2 mutants function. Differences in Cln protein abundance are unlikely to account for these results. Cln3-associated kinase is therefore likely to have an intrinsic in vivo substrate specificity distinct from that of Cln2-associated kinase, despite their functional redundancy. Consistent with the idea that Cln3 may be the primary transcriptional activator of CLN1, CLN2, and other genes, the activation of CLN2 transcription was found to be sensitive to the gene dosage of CLN3 but not to the gene dosage of CLN2.The Start transition in the Saccharomyces cerevisiae cell cycle requires the activity of one of three cyclin homologs encoded by the CLN1, CLN2, and CLN3 gene family, complexed with the cyclin-dependent kinase encoded by the CDC28 gene. Although the CLN genes are functionally redundant for cell cycle Start (39), the CLN3 gene differs sharply from the CLN1-CLN2 gene pair in structure and regulation (see reference 7 for a review). Searches for mutations resulting in lethality in strains deficient in CLN1 and CLN2 have yielded mutations in a wide range of genes, with widely varying lethal phenotypes. The general conclusion from this result has been that Cln1 and Cln2 are potent at execution of various cell biological processes associated with Start (e.g., cell polarization [4,12] and septin ring formation [4,18]), while Cln3 is relatively weak at activating these processes. Mutations in one gene (BCK2) are lethal in the absence of CLN3 but not in the absence of CLN1 and CLN2. The latter defect is significantly but not completely rescued by placing CLN2 under the control of heterologous promoters (14, 20), as Bck2 is required for efficient transcriptional activation of CLN1 and CLN2 in the absence of CLN3. This result is consistent with other data which suggest that Cln3 is the main or only physiological activator of CLN1 and CLN2 transcription (15,43,44). These r...

The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor a

Meaningful reductions in SSI can be achieved by implementing a multidisciplinary care bundle at a hospital-wide level.

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.Cyclin-dependent kinase (Cdk) activation loop phosphorylation is required for Cdk activity (9,20,39) and may be an important part of the kinase activity cycle. A glutamic acid substitution for the phosphorylated threonine in fission yeast Cdc2 has partial biological activity consistent with failure to exit from mitosis (14). In the budding yeast Cdk Cdc28p, the same mutation (T169E) resulted in a low level of Cdc28p kinase activity, correlated with a limited ability to complement the cdc28-1N allele (an allele with a G 2 -M-phase-specific defect) and no ability to complement the G 1 -S and G 2 -M-defective cdc28-4 allele (27). Although the inactivity of cdc28-T169E could indicate a requirement for a cycle of T169 phosphorylation and dephosphorylation for cell cycle control (27), the glutamic acid substitution might simply be unable to fully substitute chemically for phosphothreonine in supporting active kinase architecture (20).Here, we have used molecular evolution starting with the inactive cdc28-T169E to address two questions. First, is the combination of activation loop phosphorylation and dephosphorylation in Cdc28p of regulatory significance (as with mitogen-activated protein [MAP] kinases, for example [1,20]), or is it only a precondition for enzymatic activity that is not subject to regulatory input? If phosphorylation or dephosphorylation of this site controls Cdc28p activity in some regulatory context, then a version of Cdc28p not subject to this control should abrogate this regulation.Second, the essential gene CAK1/CIV1 encodes a kinase required for Cdc28p-T169 phosphorylation (12,21,47). Does Cak1p/Civ1p have other essential roles in addition to Cdc28p activation, such as the activation of other essential Cdk's such as Kin28 (2, 47)? MATERIALS AND METHODSCDC28...

The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT- PCR) assay. Using RT followed by nested PCR amplification for PML/RAR- alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease- free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.