Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa.

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses in Neurospora crassa. In response to light, WC-1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC-1 in dark-grown tissue and used the microarrays to identify genes regulated by an increase in WC-1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC-1 overexpression in the dark. However, only four out of 22 light-induced genes were also induced by WC-1 overexpression, demonstrating that changes in the levels of WC-1 are not sufficient to activate all light-responsive genes. The WC proteins are also required for circadian rhythms in dark-grown cultures and for light entrainment of the circadian clock, and WC-1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over-expression of WC-1 show circadian fluctuations in their levels. These data suggest that WC-1 can mediate both light and circadian responses, with an increase in WC-1 levels affecting circadian clock-responsive gene regulation and other features of WC-1, possibly its phosphorylation, affecting light-responsive gene regulation.

The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) gene.

The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day.

vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation.

con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.

Assay, isolation and characterization of circulating immune complexes from serum of gastrointestinal cancer, stage III and IV melanoma and chronic inflammatory bowel disease patients.

Circulating immune complexes (CIC) have been detected in several autoimmune diseases, and studies have also suggested that CIC provide a useful tool as tumor markers. In order to identify differences or similarities in antigenic composition, CIC from 23 patients with gastrointestinal (GI) tumors, from 20 patients with stage III and IV melanoma and from 6 patients with inflammatory bowel disease (IBD) were studied. Serum from all GI, melanoma and IBD patients showed higher levels of CIC than controls. SDS/PAGE electrophoresis under reducing conditions revealed some differences between cancer and IBD patients as far as the CIC protein composition was concerned. In melanoma patients, two fast-migrating bands, in the regions of 71-74 and 30-49 kD, were found, consistent with previously isolated and characterized antigens described in the literature.