In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arrest of cells before mitosis.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated CI-conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na + currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-l-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTRdependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na + transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.

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Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.

In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when DNA replication is blocked. In this study of budding yeast checkpoint genes, we identify and characterize another role for these checkpoint genes after DNA damagetranscriptional induction of genes. We found that three checkpoint genes (of six genes tested) have strong and distinct roles in transcriptional induction in four distinct pathways of regulation (each defined by induction of specific genes). MECI mediates the response in three transcriptional pathways, RAD53 mediates two of these pathways, and RAD17 mediates but a single pathway. The three other checkpoint genes (including RAD9) have small (twofold) but significant roles in transcriptional induction in all pathways. One of the pathways that we identify here leads to induction of MECI and RAD53 checkpoint genes themselves. This suggests a positive feedback circuit that may increase the cell's ability to respond to DNA damage. We make two primary conclusions from these studies. First, MECI appears to be the key regulator because it is required for all responses (both transcriptional and cell cycle arrest), while other genes serve only a subset of these responses. Second, the two types of responses, transcriptional induction and cell cycle arrest, appear distinct because both require MEC1 yet only cell cycle arrest requires RAD9. These and other results were used to formulate a working model of checkpoint gene function that accounts for roles of different checkpoint genes in different responses and after different types of damage. The conclusion that the yeast MECI gene is a key regulator also has implications for the role of a putative human homologue, the ATM gene.

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