Cosmids from the Vollmer-Yanofsky library identified with a chromosome VII probe.

In microorganisms, genes can often be cloned directly by complementation of mutants with a genomic library

CBM1, a Neurospora crassa genomic cosmid library in pAC3 and its use for walking on the right arm of linkage group VII

Gene cloning in Neurospora crassa is often achieved by mutant complementation. However, the cloning strategy sometimes requires the isolation of a specific genomic region (by chromosome walking) before transformation of N. crassa. This is the case, for example, if the gene to be isolated has a non-selectable phenotype. Here we specifically describe the construction of the cosmid vector, pAC3, which is designed for direct transformation of N. crassa, its utilization for the construction of a genomic library, and chromosome walking in the region of un-10 on linkage group VII

Interconnections of Reactive Oxygen Species Homeostasis and Circadian Rhythm in Neurospora crassa.

Abstract Significance: Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels. Recent Advances: The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator. Critical Issues: Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism. Future Directions: Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected. Antioxid. Redox Signal. 00, 000-000

The white collar complex is essential for sexual reproduction but dispensable for conidiation and invasive growth in Fusarium verticillioides

Fvwc1 and Fvwc2, orthologues of the wc-1 and wc-2 genes encoding for proteins of the white collar complex (WCC) in Neurospora crassa were cloned from Fusarium verticillioides and lack-of-function wc mutants were obtained by targeted gene disruption. Photo-conidiation was found to be absent in F. verticillioides, on the contrary, the wild type strain produced less conidia under continuous illumination than in the dark. Inactivation of any of the wc genes led to total female sterility, without affecting male fertility or asexual conidiation. No loss in colonization capability/invasive growth of the wc mutants was observed, when assessed on tomato fruits. Both Fvwc1 and Fvwc2 showed constitutive expression in the wild type cultures incubated in the dark and exposure to light caused only negligible increases in their transcription. Both Fvwc1 and Fvwc2 were down-regulated in a ΔFvmat1-2-1 gene disruption mutant, lacking a functional mating type (mat1-2-1) gene, suggesting that the MAT1-2-1 product has a positive regulatory effect on the white collar genes

Cross-Talk between the Cellular Redox State and the Circadian System in Neurospora

The circadian system is composed of a number of feedback loops, and multiple feedback loops in the form of oscillators help to maintain stable rhythms. The filamentous fungus Neurospora crassa exhibits a circadian rhythm during asexual spore formation (conidiation banding) and has a major feedback loop that includes the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO). A mutation in superoxide dismutase (sod)-1, an antioxidant gene, causes a robust and stable circadian rhythm compared with that of wild-type (Wt). However, the mechanisms underlying the functions of reactive oxygen species (ROS) remain unknown. Here, we show that cellular ROS concentrations change in a circadian manner (ROS oscillation), and the amplitudes of ROS oscillation increase with each cycle and then become steady (ROS homeostasis). The ROS oscillation and homeostasis are produced by the ROS-destroying catalases (CATs) and ROS-generating NADPH oxidase (NOX). cat-1 is also induced by illumination, and it reduces ROS levels. Although ROS oscillation persists in the absence of frq, wc-1 or wc-2, its homeostasis is altered. Furthermore, genetic and biochemical evidence reveals that ROS concentration regulates the transcriptional function of WCC and a higher ROS concentration enhances conidiation banding. These findings suggest that the circadian system engages in cross-talk with the cellular redox state via ROS-regulatory factors

CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components

Asymmetries in limb darkening reanalyzed

The cause of the asymmetries in limb darkening reported by Neckel and Labs (1987) is discussed on the basis of new, stray-light-free observations, and found to be of instrumental origin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43709/1/11207_2004_Article_BF00158296.pd

Genome-Wide Analysis of Light- and Temperature-Entrained Circadian Transcripts in Caenorhabditis elegans

Transcriptional profiling experiments identify light- and temperature-entrained circadian transcripts in C. elegans

Broadband Dielectric Spectroscopy on Human Blood

Dielectric spectra of human blood reveal a rich variety of dynamic processes. Achieving a better characterization and understanding of these processes not only is of academic interest but also of high relevance for medical applications as, e.g., the determination of absorption rates of electromagnetic radiation by the human body. The dielectric properties of human blood are studied using broadband dielectric spectroscopy, systematically investigating the dependence on temperature and hematocrit value. By covering a frequency range from 1 Hz to 40 GHz, information on all the typical dispersion regions of biological matter is obtained. We find no evidence for a low-frequency relaxation (alpha-relaxation) caused, e.g., by counterion diffusion effects as reported for some types of biological matter. The analysis of a strong Maxwell-Wagner relaxation arising from the polarization of the cell membranes in the 1-100 MHz region (beta-relaxation) allows for the test of model predictions and the determination of various intrinsic cell properties. In the microwave region beyond 1 GHz, the reorientational motion of water molecules in the blood plasma leads to another relaxation feature (gamma-relaxation). Between beta- and gamma-relaxation, significant dispersion is observed, which, however, can be explained by a superposition of these relaxation processes and is not due to an additional delta-relaxation often found in biological matter. Our measurements provide dielectric data on human blood of so far unsurpassed precision for a broad parameter range. All data are provided in electronic form to serve as basis for the calculation of the absorption rate of electromagnetic radiation and other medical purposes. Moreover, by investigating an exceptionally broad frequency range, valuable new information on the dynamic processes in blood is obtained.Comment: 17 pages, 9 figure