Identifying in vivo pathways using genome-wide genetic networks.

Synthetic genetic interactions occur between two genes when the double mutant displays a phenotype much more severe than does either single mutant alone. Global networks of such interactions are now being systematically determined, spearheaded by the budding yeast genome. Genetic interactions reflect in vivo relationships between gene products. Extracting that functional information from such genetic networks is now possible by exploiting and modifying the key concept of congruence. Here, we focus on synthetic genetic interactions between pairs of null mutations in non-essential yeast genes. We summarize how to identify biological pathways from these emerging networks, using illustrative examples.

Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomycescerevisiae.

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3-2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3-1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants.

BACKGROUND: The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan. Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function. RESULTS: Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation. CONCLUSIONS: The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation.

Midwifery triage of first trimester bleeding.

Approximately one of five pregnant women will experience bleeding during the first trimester of pregnancy. Of these women, about half will go on to have a spontaneous abortion. Comfort with triaging this common problem assists the midwife in providing quality, cost-effective care without eliminating continuity. This article reviews the causes of early pregnancy bleeding, offers strategies to differentiate between these causes, and identifies emergent from nonemergent presentations. Triage and management strategies for women with an impending or threatened spontaneous abortion are reviewed, exploring the alternative of expectant management.

Extending ventilator circuit change interval beyond 2 days reduces the likelihood of ventilator-associated pneumonia.

OBJECTIVE: To determine the risk of acquiring ventilator-associated pneumonia (VAP) and the impact on costs when extending ventilator circuit change intervals beyond 2 days to 7 and 30 days. DESIGN: Prospective 4-year review of mechanically ventilated patients. SETTING: The respiratory and medical ICUs of an 800-bed tertiary teaching Veterans Affairs hospital. PATIENTS: All adult patients receiving mechanical ventilation from January 1991 through December 1994. INTERVENTIONS: Ventilator circuits with active heated water humidifiers were changed at 2-day intervals during a 2-year control period, followed by 7-day and 30-day intervals (for 1 year each). Heated wire circuits were adopted with the 30-day interval. The rate of VAP per 1,000 ventilator days was calculated for each circuit change interval group. Survival analysis was used to model VAP with ventilator circuit change to determine risk. RESULTS: During the study period, 637 patients received mechanical ventilation. During the 2 years with 2-day change intervals, the VAP per 1,000 ventilator days was 11.88 (n=343), compared with 3.34 (n=137) and 6.28 (n=157) for 7-day and 30-day change intervals, respectively. The risk of acquiring a VAP for those with a circuit change every 2 days was significantly greater (relative risk, 3.1; p=0.0004; 95% confidence interval, 1.662, 5.812) than those with the 7- and 30-day circuit changes. Extending circuit change intervals reduced supply and labor costs averaging $4,231/yr for each ventilator in use. CONCLUSIONS: Circuit change intervals of 7 and 30 days have lower risks for VAP than the 2-day intervals, yielding substantial reductions in morbidity as well labor and supply costs.