Stopping time: the genetics of fly and mouse circadian clocks.

Forward genetic analyses in flies and mice have uncovered conserved transcriptional feedback loops at the heart of circadian pacemakers. Conserved mechanisms of posttranslational regulation, most notably phosphorylation, appear to be important for timing feedback. Transcript analyses have indicated that circadian clocks are not restricted to neurons but are found in several tissues. Comparisons between flies and mice highlight important differences in molecular circuitry and circadian organization. Future studies of pacemaker mechanisms and their control of physiology and behavior will likely continue to rely on forward genetics.

takeout, a novel Drosophila gene under circadian clock transcriptional regulation.

We report the identification and characterization of a new Drosophila clock-regulated gene, takeout (to). to is a member of a novel gene family and is implicated in circadian control of feeding behavior. Its gene expression is down-regulated in all of the clock mutants tested. In wild-type flies, to mRNA exhibits daily cycling expression but with a novel phase, delayed relative to those of the better-characterized clock mRNAs, period and timeless. The E-box-containing sequence in the to promoter shows impressive similarities with those of period and timeless. However, our results suggest that the E box is not involved in the amplitude and phase of the transcriptional cycling of to. The circadian delayed transcriptional phase is therefore most likely the result of indirect regulation through unknown transcription factors.

Appropriateness of prostate-specific antigen testing.

We established criteria for appropriate use of the prostate-specific antigen (PSA) assay and used them to evaluate PSA test utilization at 1 tertiary care institution. During a 6-month period, 2,330 PSA results were reported for outpatients and 95 for inpatients. We reviewed medical records for a random sample of 338 outpatient results (14.51%) and all 95 inpatient results, of which 21% (71/338) of outpatient and 17% (16/95) of inpatient results were inappropriate according to our test utilization criteria. Among outpatients, 52% of tests were done for screening and 19% for monitoring for cancer recurrence. For inpatients, workup for cancer (53/95 [56%]) was the most frequent indication for testing and screening the second (24/95 [25%]). Of tests failing the criteria, 66 (76%) of 87 resulted from excessively frequent and age-inappropriate screening. We assessed the potential effect on clinical outcome if these tests were not performed. Of the 87 tests considered inappropriate, only 1 test result influenced clinical management for patients younger than 75 years. By instituting simple limits on age and frequency, we estimate that 74% (64/87) of the inappropriate tests could have been eliminated.

Biological clocks.

Circadian rhythms describe biological phenomena that oscillate with an approximately 24-hour cycle. These rhythms include blood pressure, body temperature, hormone levels, the number of immune cells in blood, and the sleep-wake cycle. In this paper, we will focus on common genes between species that are responsible for determining the circadian behavior, especially some transcription factors (i.e., switch genes) that serve to regulate many circadian rhythm genes. The intent of this summary is to introduce the common molecular mechanism of biological clocks between flies and humans and then to describe the research from three laboratories that was presented in the session.

A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless.

We report the identification, characterization, and cloning of a novel Drosophila circadian rhythm gene, dClock. The mutant, initially called Jrk, manifests dominant effects: heterozygous flies have a period alteration and half are arrhythmic, while homozygous flies are uniformly arrhythmic. Furthermore, these flies express low levels of the two clock proteins, PERIOD (PER) and TIMELESS (TIM), due to low per and tim transcription. Mapping and cloning of the Jrk gene indicates that it encodes the Drosophila homolog of mouse Clock. The mutant phenotype results from a premature stop codon that eliminates much of the putative activation domain of this bHLH-PAS transcription factor, thus explaining the dominant features of Jrk. The remarkable sequence conservation strongly supports common clock components present in the common ancestor of Drosophila and mammals.

The C-terminal region of Drosophila heat shock factor (HSF) contains a constitutively functional transactivation domain.

The heat shock transcription factor (HSF) is constitutively expressed in Drosophila cells as an inactive monomer. Upon heat shock HSF undergoes trimerization and acquires high affinity DNA binding ability leading to specific interaction with its cognate elements in heat shock promoters. Here we show that the transactivation function of HSF is conferred by the extreme C-terminal region of the protein. Deletion analysis of HSF fragments fused to the GAL4 DNA-binding domain demonstrates that transactivation is dependent on HSF residues 610-691. This domain is located beyond the C-terminal heptad repeat (leucine zipper 4) whose presence or integrity is dispensable for transactivation. The transactivation domain is functional in the absence of heat shock and can be replaced by the extreme C-terminal region of human HSF1. The Drosophila and human HSF transactivation domains are both rich in hydrophobic and acidic residues and may be structurally conserved, despite limited sequence identity.

Drosophila melanogaster as a model for study of general anesthesia: the quantitative response to clinical anesthetics and alkanes.

Using an assay of the geotactic behavior of the fruit fly, we have quantified anesthetic effects in Drosophila melanogaster. We determined dose-responses for nine conventional anesthetics and a series of n-alkanes. For the conventional anesthetics, anesthetic potency and olive oil/gas partition coefficients are well correlated. However, as one ascends the series of alkanes of chain lengths 5-11, the increase in anesthetic potency is considerably less than that predicted by olive oil solubility. These examples of agreement and disagreement with the Meyer-Overton rule provide a basis for comparing the responses of fruit flies with those of higher organisms; such comparisons suggest that the anesthetic target is evolutionarily conserved. For alkanes with chain lengths greater than 11, anesthetic effects are variable and slow to develop. This behavior reflects limited volatility and provides no evidence for a cut-off in the action of alkanes at their target.

Intermittent urethral catheterization in the elderly.

Thirty-five elderly patients receiving intermittent urethral catheterization in a Veterans Administration Hospital and attached nursing home care unit were prospectively studied for development of bacteriuria and/or urinary tract infection. Thirty-one of the 35 patients (88.6%) developed urinary tract colonization. The mean time from initiation of catheterization to development of colonization was 5.7 +/- 1.3 days. Persistent bacteriuria with one or several different microorganisms developed in 17 patients. The most common colonizing organisms were coagulase negative staphylococci, Klebsiella pneumoniae, and enterococcus. Four patients (11%) developed symptomatic urinary tract infection. Although urinary tract colonization was common in patients receiving intermittent urethral catheterization, especially in those with poor functional status, infection was uncommon. Based on these results, intermittent urethral catheterization appears to be a safe and effective method of bladder drainage in elderly male patients when performed with sterile techniques over short periods of time in the nursing home or hospital setting.