Involvement of a G Protein Regulatory Circuit in Alternative Oxidase Production in Neurospora crassa.

The Neurospora crassa nuclear aod-1 gene encodes an alternative oxidase that functions in mitochondria. The enzyme provides a branch from the standard electron transport chain by transferring electrons directly from ubiquinol to oxygen. In standard laboratory strains, aod-1 is transcribed at very low levels under normal growth conditions. However, if the standard electron transport chain is disrupted, aod-1 mRNA expression is induced and the AOD1 protein is produced. We previously identified a strain of N. crassa, that produces high levels of aod-1 transcript under non-inducing conditions. Here we have crossed this strain to a standard lab strain and determined the genomic sequences of the parents and several progeny. Analysis of the sequence data and the levels of aod-1 mRNA in uninduced cultures revealed that a frameshift mutation in the flbA gene results in the high uninduced expression of aod-1 The flbA gene encodes a regulator of G protein signaling that decreases the activity of the Gα subunit of heterotrimeric G proteins. Our data suggest that strains with a functional flbA gene prevent uninduced expression of aod-1 by inactivating a G protein signaling pathway, and that this pathway is activated in cells grown under conditions that induce aod-1 Induced cells with a deletion of the gene encoding the Gα protein still have a partial increase in aod-1 mRNA levels, suggesting a second pathway for inducing transcription of the gene in N. crassa We also present evidence that a translational control mechanism prevents production of AOD1 protein in uninduced cultures.

Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism.

In Neurospora crassa, blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa, we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome.

The characteristics of sexual behavior and extent of condom usage among sexually active Croatians from Eastern Croatia.

OBJECTIVE: To determine the extent of condom usage as a method of protection from sexually transmitted infections (STIs) among sexually active individuals obliged to present for periodical health examination at the Institute of Public Health for the Osijek-Baranya County, in Osijek, eastern Croatia. MATERIAL AND METHODS: During February 2004, a cross-sectional questionnaire survey was conducted. The research tool was an unidentified 20-item questionnaire addressing the sexual behavior of study subjects and their partners and methods of protection from STIs. Descriptive statistics and χ(2) - test were used for data analysis. RESULTS: The response rate was 84.2% (278/330). A total of 278 subjects, 96 (34.5%) males and 182 (65.5%) females, 167 (60.1%) married and 111 (39.9%) single, mean age 31.3±8.4, age range 18-52 years were enrolled in the study. Study results revealed 22.3% (62/278) subjects to have had two or more sexual partners over the one-year period and the use of condom was reported by 40.3% (25/62) of those subjects. CONCLUSIONS: The obtained results point to the need for additional education on protection from sexually transmitted infections and on risky sexual behavior, with special reference to the role of condom usage in the prevention of these diseases and their detrimental effects on the reproductive health of sexually active individuals.