Carlene Allen Raper 1925-2019

The field of fungal genetics and biology lost one of its founding anchors with the death of Carlene Raper on September 5, 201

A recommendation for naming proteins in Neurospora

The issue of gene product names is important in as much as it promotes consistency within the literature and promotes accessibility of the Neurospora literature to readers more familiar with other organisms

Phosphorylation of the Neurospora Clock Protein FREQUENCY Determines its Degradation Rate and Strongly Influences the Period Length of the Circadian Clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (\u3e30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock

Monitored Geologic Repository Operations Monitoring and Control System Description Document

The Monitored Geologic Repository Operations Monitoring and Control System provides supervisory control, monitoring, and selected remote control of primary and secondary repository operations. Primary repository operations consist of both surface and subsurface activities relating to high-level waste receipt, preparation, and emplacement. Secondary repository operations consist of support operations for waste handling and treatment, utilities, subsurface construction, and other selected ancillary activities. Remote control of the subsurface emplacement operations, as well as, repository performance confirmation operations are the direct responsibility of the system. In addition, the system monitors parameters such as radiological data, air quality data, fire detection status, meteorological conditions, unauthorized access, and abnormal operating conditions, to ensure a safe workplace for personnel. Parameters are displayed in a real-time manner to human operators regarding surface and subsurface conditions. The system performs supervisory monitoring and control for both important to safety and non-safety systems. The system provides repository operational information, alarm capability, and human operator response messages during emergency response situations. The system also includes logic control to place equipment, systems, and utilities in a safe operational mode or complete shutdown during emergency response situations. The system initiates alarms and provides operational data to enable appropriate actions at the local level in support of emergency response, radiological protection response, evacuation, and underground rescue. The system provides data communications, data processing, managerial reports, data storage, and data analysis. This system's primary surface and subsurface operator consoles, for both supervisory and remote control activities, will be located in a Central Control Center (CCC) inside one of the surface facility buildings. The system consists of instrument and control equipment and components necessary to provide human operators with sufficient information to monitor and control the operation of the repository in an efficient and safe manner. The system consists of operator consoles and workstations, multiple video display terminals, communications and interfacing equipment, and instrument and control software with customized configuration to meet the needs of the Monitored Geologic Repository (MGR). Process and logic controllers and the associated input/output units of each system interfaced with this system will be configured into Remote Terminal Units (RTU) and located close to the systems to be monitored and controlled. The RTUs are configured to remain operational should communication with CCC operations be lost. The system provides closed circuit television to selectively view systems, operations, and equipment areas and to aid in the operation of mechanical systems. Control and monitoring of site utility systems will be located in the CCC. Site utilities include heating, ventilation, and air conditioning equipment; plant compressed air; plant water; firewater; electrical systems; and inert gases, such as nitrogen, if required. This system interfaces with surface and subsurface systems that either generate output data or require remote control input. The system interfaces with the Site Communications System for bulk storage of operational data, on-site and off-site communication, and a plant-wide public announcement system. The system interfaces with the Safeguards and Security System to provide operational status and emergency alarm indications. The system interfaces with the Site Operation System to provide site wide acquisition of data for analysis and reports, historical information for trends, utility information for plant operation, and to receive operating plans and procedures

The Clock affecting 1 mutation of Neurospora is a recurrence of the frq\u3csup\u3e7\u3c/sup\u3e mutation7

The clock affecting-1 (cla-1) mutation of Neurospora crassa increases the period and decreases temperature compensation of the circadian rhythm, and was thought to define an uncloned gene with a possible role in the Neurospora clock. This defect, thought to be due to a translocation, was associated with a slow growth rate and a period of about 27 h at 25cla-1 and found the growth rate and period defects to be due to linked independent mutations. The translocation was not the cause of the long period. The csp-1 mutation, present in the original cla-1 strain, had a period shortening effect, thus cla-1 strains lacking csp-1 had a period length similar to that of frequency7 (frq7). The cla-1 period defect mapped to the frq locus, and sequencing of frq revealed cla-1 to be a re-isolation of frq7

Temperature-Sensitive and Circadian Oscillators of Neurospora crassa Share Components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria Macrospora Provides New Insights into Essential and Nonessential Sexual Regulators

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes

How Temperature Changes Reset a Circadian Oscillator

Circadian rhythms control many physiological activities. The environmental entrainment of rhythms involves the immediate responses of clock components. Levels of the clock protein FRQ were measured in Neurospora at various temperatures; at higher temperatures, the amount of FRQ oscillated around higher levels. Absolute FRQ amounts thus identified different times at different temperatures, so temperature shifts corresponded to shifts in clock time without immediate synthesis or turnover of components. Moderate temperature changes could dominate light-to-dark shifts in the influence of circadian timing. Temperature regulation of clock components could explain temperature resetting of rhythms and how single transitions can initiate rhythmicity from characteristic circadian phases

DISPOSAL CONTAINER HANDLING SYSTEM DESCRIPTION DOCUMENT

The Disposal Container Handling System receives and prepares new disposal containers (DCs) and transfers them to the Assembly Transfer System (ATS) or Canister Transfer System (CTS) for loading. The system receives the loaded DCs from ATS or CTS and welds the lids. When the welds are accepted the DCs are termed waste packages (WPs). The system may stage the WP for later transfer or transfer the WP directly to the Waste Emplacement/Retrieval System. The system can also transfer DCs/WPs to/from the Waste Package Remediation System. The Disposal Container Handling System begins with new DC preparation, which includes installing collars, tilting the DC upright, and outfitting the container for the specific fuel it is to receive. DCs and their lids are staged in the receipt area for transfer to the needed location. When called for, a DC is put on a cart and sent through an airlock into a hot cell. From this point on, all processes are done remotely. The DC transfer operation moves the DC to the ATS or CTS for loading and then receives the DC for welding. The DC welding operation receives loaded DCs directly from the waste handling lines or from interim lag storage for welding of the lids. The welding operation includes mounting the DC on a turntable, removing lid seals, and installing and welding the inner and outer lids. After the weld process and non-destructive examination are successfully completed, the WP is either staged or transferred to a tilting station. At the tilting station, the WP is tilted horizontally onto a cart and the collars removed. The cart is taken through an air lock where the WP is lifted, surveyed, decontaminated if required, and then moved into the Waste Emplacement/Retrieval System. DCs that do not meet the welding non-destructive examination criteria are transferred to the Waste Package Remediation System for weld preparation or removal of the lids. The Disposal Container Handling System is contained within the Waste Handling Building System. This includes the primary hot cell bounded by the receiving area and WP transport exit air locks; and isolation doors at ATS, CTS, and Waste Package Remediation. The hot cell includes areas for welding, various staging, tilting, and WP transporter loading. There are associated operating galleries and equipment maintenance areas outside the hot cell. These areas operate concurrently to accommodate the DC/WP throughput rates and support system maintenance. The new DC preparation area is located in an unshielded structure. The handling equipment includes DC/WP bridge cranes, tilting stations, and horizontal transfer carts. The welding area includes DC/WP welders and staging stations. Welding operations are supported by remotely operated equipment including a bridge crane and hoists, welder jib cranes, welding turntables, and manipulators. WP transfer includes a transfer/decontamination and transporter load area. The transfer operations are supported by a remotely operated horizontal lifting system, decontamination system, decontamination and inspection manipulator, and a WP horizontal transfer cart. All handling operations are supported by a suite of fixtures including collars, yokes, lift beams, and lid attachments. Remote equipment is designed to facilitate decontamination and maintenance. Interchangeable components are provided where appropriate. Set-aside areas are included, as required, for fixtures and tooling to support off-normal and recovery operations. Semi-automatic, manual, and backup control methods support normal, maintenance, and recovery operations. The system interfaces with the ATS and CTS to provide empty and receive loaded DCs. The Waste Emplacement/Retrieval System interfaces are for loading/unloading WPs on/from the transporter. The system also interfaces with the Waste Package Remediation System for DC/WP repair. The system is housed, shielded, supported, and has ventilation boundaries by the Waste Handling Building (WHB). The system is ventilated by the WHB Ventilation System, which in conjunction with ventilation boundaries ensure that airborne radioactivity is confined by high efficiency particulate air filtration units. Electrical power is supplied by the Waste Handling Building Electrical System. The system also interfaces with each of the DC systems. New DCs are received from a commercial transport system

Alternative Use of DNA Binding Domains by the Neurospora White Collar Complex Dictates Circadian Regulation and Light Responses

In the Neurospora circadian system, the White Collar complex (WCC) of WC-1 and WC-2 drives transcription of the circadian pacemaker gene frequency (frq), whose gene product, FRQ, as a part of the FRQ-FRH complex (FFC), inhibits its own expression. The WCC is also the principal Neurospora photoreceptor; WCC-mediated light induction of frq resets the clock, and all acute light induction is triggered by WCC binding to promoters of light-induced genes. However, not all acutely light-induced genes are also clock regulated, and conversely, not all clock-regulated direct targets of WCC are light induced; the structural determinants governing the shift from WCC\u27s dark circadian role to its light activation role are poorly described. We report that the DBD region (named for being defective in binding DNA), a basic region in WC-1 proximal to the DNA-binding zinc finger (ZnF) whose function was previously ascribed to nuclear localization, instead plays multiple essential roles assisting in DNA binding and mediating interactions with the FFC. DNA binding for light induction by the WCC requires only WC-2, whereas DNA binding for circadian functions requires WC-2 as well as the ZnF and DBD motif of WC-1. The data suggest a means by which alterations in the tertiary and quaternary structures of the WCC can lead to its distinct functions in the dark and in the light