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Position Summary
Plan, coordinate, direct, and supervise all operations and programs relating to the City's potable water supply, watershed, and water quality laboratory in a safe, continuous, and cost effective manner, in compliance with local, State and federal rules and regulations. These tasks are illustrative only and may include other related duties.
Essential Functions
Plans, directs, and monitors the City's potable water treatment operations, watershed and water quality laboratory for continuous, safe and cost effective operation. Manages and maintains operating facilities and equipment as the Oregon Health Authority designated direct responsible person in charge of treatment processes. Assures treatment process adheres to established Oregon Health Department and EPA regulations, and maintains required documentation and records. Develops goals, objectives, policies and maintenance programs to minimize equipment downtime and service interruptions. Utilizes staff efficiently and effectively to cover facility operations and meet daily demands. Develops, implements and monitors operational and maintenance work plans, processes, and procedures to achieve work group objectives and performance measures. Maintains knowledge base to communicate, administer, and ensure compliance with all State and federal programs and regulations related to the Safe Drinking Water Act, OSHA, ADA and all other applicable rules and laws. Prepares and submits necessary monitoring information to appropriate State and federal agencies for compliance with regulations. Manages watershed to ensure water quality in compliance with all applicable regulations and quantity to meet the service needs of the community. Maintains required documentation and records. Evaluates effectiveness of services and programs, and recommends improvements and modifications. Researches regulatory developments and formulates strategies to meet operational and maintenance requirements. Provides quality assurance management. Forecasts and plans for short- and long-range operations and maintenance needs. Participates in design review, to include equipment evaluation, research and recommendation of improved process design. Plans, coordinates, manages and/or participates in technical projects related to areas of responsibility. Develops and implements contract documents, plans, specifications, special projects, and bid documents. Conducts cost analysis on purchasing and contracting alternatives. Prepares purchase orders. Administers contracts and manages budgets associated with contract work. Communicates and collaborates with consultants and other organization staff working on projects. Resolves performance issues with contractors. Writes letters, memorandums, technical documents, and reports related to the operation, strategic planning, and management of areas of responsibility. Develops or directs the development of technical information resources, project data and historical reference files. Develops, managers, and monitors the budget for the assigned budget area. Establishes and monitors appropriate department financial measures. Interprets and uses financial data to make informed business decisions. Monitors and coordinates purchasing of materials, equipment, and supplies to comply with purchasing policy guidelines and to remain within established budgetary constraints. Provides expert consultation, assistance, technical advice, and support to the Division Manager. Participates in division planning and coordination. Functions as a team member in the management and operation of assigned program areas. Provides technical and operational input for improvements to assigned program areas; provides technical guidance to ensure highly productive and effective work teams and programs. Coordinates activities with other supervisors, managers and work groups. Demonstrates understanding and good judgment in the evaluation of complex technical and/or financial information and alternatives related to program and policy development. Responds to emergency calls during business hours and after hours, and coordinates emergency response activities. Must be able to respond to calls within 30 minutes.If situation warrants it, must be able to respond on site within 60 minutes. Maintains training and expertise in National Incident Management System (NIMS).Participates in emergency management operations (EOC).Mobilizes staff and resources to address emergency response efforts. Effectively promotes and supervises a safe work environment. Complies with all safety rules and performs work in a safe manner. Ensures staff maintains a safe working environment in compliance with federal, State, and City health and safety guidelines and regulations. Manages and evaluates safety, training, and security policies and procedures.Investigates accidents and claims and implements modifications as required. Conducts staff safety meetings and ensures safety inspections are conducted as required. Effectively supervises assigned staff.Performs personnel duties such as selection, coaching/mentoring, training, employee/labor relations, evaluating performance, succession planning, discipline, and termination. Creates and maintains an effective work environment by communicating goals and expectations for staff performance, counseling employees as appropriate, and supporting employee engagement efforts. Communicates with the public, local officials, other municipalities, and other City departments on matters related to the water treatment facility. Develops and provides public information and educational services. Participates in and represents the City at technical conferences, task force meetings, professional organizations, and public meetings. Serves as a liaison to private contractors, engineers, property owners, and regulating agencies in matters related to operation of a water treatment facility. Answers questions and provides information to the public; investigates complaints and recommends corrective action as necessary to resolve complaints. Attends meetings and/or serves on a variety of City committees and task teams. Attends necessary training to maintain state certification levels and City safety standards. Acts ethically and honestly; applies ethical standards of behavior to daily work activities and interactions. Builds confidence in the City through own actions. Complies with all Administrative Policies.Performs work in accordance with Council Policies and Municipal Code sections applicable to the position. Represents the City of Corvallis by responding to the public, community members, its employees, and others in a prompt, professional, and courteous manner.Regards everyone, internal and external, as a customer and delivers the best service possible in a respectful and patient manner.Maintains effective work relationships. Arrives to work, meetings and other work-related functions on time and maintains regular job attendance. Operates and drives a motor vehicle and/or truck safely and legally.
Qualifications and Requirements
Education and ExperienceAssociate of Applied Science degree in water/wastewater technology or two-year degree from an accredited college or university in Environmental Science, Business Administration, or a related field and at least seven years of professional experience implementing or managing the operations and maintenance of a conventional surface water treatment plant. ORBachelor's degree from an accredited college or university in Business Administration, Civil or Environmental Engineering, Operations Management, biology, chemistry or related field and at least three years of professional experience implementing or managing the operations and maintenance of an conventional surface water treatment plant.At least three years of work experience in an increasingly responsible supervisory or leadership capacity.Experience working in the public sector or a government agency preferred.Knowledge, Skills and AbilitiesHigh level of technical expertise in water treatment/water quality and a broad knowledge of water utility operations.Knowledge of modern principles and practice of a public water treatment plant operation, and maintenance methods, materials, and equipment used in treatment plants.Knowledge of the federal Safe Drinking Water Act, the National Pollutant Discharge Elimination System, Oregon Health Department and other applicable rules and regulations pertaining to potable water and watersheds, and their relationship to the water utility. Ability to interpret and apply applicable local, State and federal regulatory requirements.Knowledge of principles of supervision and personnel practices, and budgeting and recordkeeping.Ability to work under time constraints, manage evolving situations, perform multiple tasks effectively, evaluate and motivate staff to maximize organizational efficiency and effectiveness.Ability to read plans, specifications, engineering drawings and interpret maps. Ability to develop and implement operations and maintenance management programs and procedures.Strong leadership, communication, and organizational skills. Ability to plan, assign, and supervise the work of subordinates to accomplish goals and objectives. Effective written and oral communication skills; ability to prepare and present comprehensive written and oral reports on technical and complex issues to a non-technical audience.Ability to make appropriate decisions, apply independent judgment, solve complex problems and prioritize support activities and programs in a cost effective manner.Get along well with coworkers and maintain working relationships, including effective labor relations.Special RequirementsState of Oregon Water Treatment Grade Level IV certification required at time of hire.State of Oregon filtration endorsement within one (1) year of appointment.Maintenance of First Aid and CPR certification.Possession or ability to obtain and maintain a valid Oregon Drivers License.Respond to emergency calls during business hours and after hour call outs. Must be able to respond to calls within 30 minutes. If the situation warrants it, must be able to respond on site within 60 minutes prior to the end of probationary period.Occasional travel includes local and regional meetings.This position may regularly be required to work evenings and weekends.Demonstrable commitment to equity, diversity and inclusion; ability to promote equity, diversity and inclusion principles with employees in day to day operations. Ability to successfully use equity, diversity and inclusion principles within the department and organization-wide.Demonstrable commitment to, and understanding of, sustainability and the ability to promote commitment, understanding, and use of sustainability principles by employees for day to day operations required. Ability to hold employees accountable for sustainability objectives.The individual shall not pose a direct threat to the health or safety of the individual or others in the workplace.
Additional Information
SUPERVISORY OR LEAD WORK RESPONSIBILITIESThis position performs personnel duties such as selection, coaching/mentoring, training, employee/labor relations, evaluating performance, succession planning, discipline and termination.Creates and maintains an effective work environment by communicating goals and expectations for staff performance; counseling employees as appropriate, and supporting employee engagement efforts. PHYSICAL REQUIREMENTSIn the performance of job duties, the employee is frequently required to sit, walk, stand and use a computer for extensive periods of time; occasional grasping, reaching, and turning; frequent hearing and talking in person and on the phone. Required to bend, squat, and negotiate different terrains when in the field. The employee must be able to hear changes in frequencies and identify abnormal noises, and have the ability to smell chlorine and/or other chemicals used in the treatment process. Manual dexterity and coordination required to operate computer, plant and office equipment. TOOLS AND EQUIPMENT USEDIn the performance of job duties, the incumbent may use the following tools and/or equipment: multi-line phone system; personal computer including SCADA, Distributive Control System, word processing, spreadsheet, and data base software; copy and fax machines; calculator; automobile or truck and other related tools and equipment. WORK ENVIRONMENTThe work performed in an office environment is usual office working conditions. The noise level in the work area is typical of most office environments, with some telephones, personal interruptions and background noises. Field work includes exposure to a variety of hazardous situations on a recurring basis including adverse weather conditions, production equipment, loud noise, confined spaces, hazardous chemicals, environmental hazards, live utilities and air borne hazards.


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What we do
Raspberry Pi-based training in Computer Science
Computer Science describes how the various elements of computers are designed, how they work, and how they may be programmed.
The Raspberry Pi, a low-cost micro-computer about the size of a credit card, is considered to be an ideal platform for teaching young individuals, as well as adults how computers work.
The Raspberry Pi is the brainchild of the Raspberry Pi Foundation, a UK based charity which is supported by the University of Cambridge Computer Laboratory and Broadcom.
The Raspberry Pi enables individuals to learn how to program, work with electronics and computer hardware, as well as interact directly with the physical world through technology.
Computer Network Training
Our foundational training in computer networking will also be based on the Raspberry Pi and other open source networking solutions. The training is based on practical activities and will also show students how to control physical hardware over a network.
Why we do this
We are aware that our students and other beneficiaries may just need a helping hand along the way to a successful career. So we would like to pass on our experience in computer science and networking to these individuals; and hope that they in future pass it forward too.
Training for Social Impact
There is sufficient evidence about the low prospects of rewarding employment opportunities for young people from disadvantaged backgrounds, living in Glasgow. These individuals have a higher possibility of social exclusion or marginalisation from society resulting in long term unemployment and difficult integration into local communities. Networks4Learning seeks to make a difference for these individuals as beneficiaries of the project, with a focus on the BME community.
Learner Centred Training
Networks4Learning provides specialized information technology training in the field of computer networking. Our training approach is learner centred, informal and tailored to meet our beneficiaries' unique learning styles.
We provide training materials, have networking lab equipment, and an environment to ensure the best possible learning outcome.

subset: commoncrawl


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Coronation Street star Michelle Collins has named Pat Phoenix's Elsie Tanner as her all-time favourite TV character.
The Weatherfield actress, who plays Rovers Return landlady Stella Price, described Corrie as a "female show" in an interview with Metro.
"It's a strong female lead in a female show. Corrie is very matriarchal; the women are always painted as strong, while a lot of the men are quite hapless and a bit weak," she said.
"Elsie was one of the original female icons that came out of the kitchen-sink drama. Life was hard for her, she always had broken relationships but she was gutsy and feisty."
Collins added: "I hate the phrase but she was a 'tart with a heart'."
The 51-year-old, who formerly starred in EastEnders, also reserved praise for US sitcoms Modern Family and The Big Bang Theory.
Last week, a report emerged that Collins's Corrie character Stella will embark on a fling with Jason Grimshaw.


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Recent Posts
Category Archives: Technology
The Chicago Tribune: okay, the website makes up for it
Every day, millions of people rely on local news sites, stations, papers, and radio stations to update them on the important events occurring around the world. In a huge metropolis such as Chicago, journalists have quite a large task before … Continue reading
Posted in Education, Entertainment, Journalism, News, Opinion, Technology Tagged chicago, demographics, facebook, google+, mobile, pay, retweet, tribune, wall, website Leave a comment
After this post, you're gonna have to pay me.
Just kidding. But not all websites are. As a student majoring in journalism, I've heard the pros and cons of paywalls on news sites weighed more times than I can count. I think it's safe to say that almost everyone … Continue reading
Posted in Education, Entertainment, Journalism, News, Opinion, Technology Tagged advertisements, CPM, decrease, journalism, journalist, pay, pay-per-click, paywall, wall 2 Comments
Map of Crime in Whitewater, WI
Posted in Crime Map, Education, News, Student Life, Technology Tagged crime, dui, intoxication, theft, whitewater, wisconsin Leave a comment
Romney's "private" comments go public
We journalists know that, when you're in the public eye, everything you do can and will be watched. You're fair game. …Unless it's a private event. Right, Romney? At a "private" fundraiser on May 17th held in the home of Marc Leder, … Continue reading
Posted in Education, Journalism, News, Opinion, Politics, Technology Tagged 47%, barack, fundraiser, jones, leder, marc, marc leder, mitt, mitt romney, mother, motherjones, obama, private, reaction, romney, transcripts, victims, videos Leave a comment
iPhone 5 vs. Samsung Galaxy S3 Drop Test
For those of you who are curious which phone can better withstand your possible abuse, check out this informational video.
Video September 26, 2012 Tagged broken, crack, drop, iphone, samsung, screen, technology Leave a comment
More skill sets needed for new jobs
In Thomas Friedman's column "New Rules," Friedman talks about children in Estonia who are being taught computer programming in schools beginning at age seven and ending at age 16. Friedman talks not so much about the fact that children are … Continue reading
Posted in Education, Journalism, News, Opinion, Technology Tagged computer, estonia, Friedman, new, programming, rules, Thomas Leave a comment


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Quick Kickin Ketchup
At Fitter Food we LOVE ketchup but unfortunately many out there are full of added sugar which we try to avoid as often as we can BUT that does not mean we can't have it, we just simply make our own. Check out this smokey twist we made recently. Tastes fantastic and is so easy to make, you can whack it together whilst dinner is in the oven. You can keep this in the fridge for 3-4 days in an air tight container.View Recipe


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© 2019 All rights Reserved. Digital by DR.SEO LLC
Maintaining good dental health is important in preventing dental illnesses that are quite common. Most people who go for dental treatment usually have a dental infection that relates to poor dental hygiene. While it is recommended that you brush and floss your teeth regularly, this may not be the only way to maintain dental health.
Regular visits to the dentist can help improve your dental health and in turn avoid any dental infections and illnesses. In the event of dental infection, dental restorations Westwood can be done to restore the state of your teeth and gums.
Visit Cosmetic Dental of Westwood today and enjoy top-notch dental restoration services.
Missing teeth tend to affect one's general smile. Aside from this, a missing tooth can as well cause other orthodontic issues that may become quite difficult to treat.
One of them includes tooth misalignment. Once a single tooth is removed, the adjacent teeth may begin shifting their position to fill the gap thus changing their general alignment and symmetry with upper or lower teeth.
In such a case, one may develop biting conditions that may interfere with tooth function.
Dental restorations Westwood can be used in filling gaps left by missing teeth thus preventing shifting of teeth.
Tooth decay is a notorious dental condition that can cause teeth to become dysfunctional.
For most people, they do not realize that they have tooth decay until it is too late and the decay has spread to adjacent teeth.
Dental restorations Westwood can be used in preventing the spread of tooth decay. After visiting the dentist for tooth decay treatment, he flushes out all the bacteria and foreign material in order to leave the tooth free of decay.
After that, the dentist will use dental restoration to cover the tooth and conceal any holes or crevices that the decay can access again. In this way, tooth decay can longer affect the treated tooth.
Dental restorations can help reinforce weak and sensitive teeth. For some people, their teeth may be weak or sensitive due to an eroded enamel.
This may cause drinking hot or cold substances to be a bother since it may cause a lot of pain. Visiting a dentist for dental restoration can help reduce this issue and make your teeth stronger against external factors.
A dental restoration can be placed over the tooth to increase its strength and also ensure that the tooth is no longer affected by harsh conditions.
Visit Cosmetic Dental of Westwood today and get some of the best dental restoration services.
Gaps between your teeth can affect your smile. Through dental restoration, you can forget about these gaps since the dentist can bridge them using a special dental paste.
All the dentist will have to do is prepare a small amount of dental paste and place it on the gap.
After the paste hardens, the dentist will then shade the paste to match the color of adjacent teeth.


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Gorgeous contemporary stacking ring set with a velvety dark brown, rose cut sapphire in a pear shape. The sapphire is teamed up with freshwater pearls and a plain faceted silver band. Choose if you would like the white or mauve pearl ring along with the sapphire ring and the faceted band.
This ring stack is a UK size M (US size 6 1/4) and is ready to ship now. If you prefer a different combination of stones and pearls, or need a different size, please contact me to talk about a possible commission of your own stacking ring set.
The silver has been given a brushed finish which tones beautifully with the sapphire and the pearls, and results in a modern, yet understated set of rings, perfect for use anytime.
The sapphire measures approximately 13 mm long and 9 mm wide. The mauve pearl is 4 mm in diameter and the white one 5 mm. The bands are all made from 2.5mm silver stock.
At the moment this ring stack is not hallmarked, but that can be done before shipping, at no extra cost.


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Residential Fences
At Broken Willow we have been building quality long lasting residential fences for many years. In the past this was a service to our regular tree customers only and we didn't advertise building fences. This part of the business grew to the point that we decided to go ahead and offer it along with our other services.
We build both residential chain link and wood fences.
Our emphasis is on good looking quality fences that are cost effective because they last a very long time.
Our wood fences are built with all cedar lumber on metal posts.
Our chain link fences are built with longer heavier posts.
We can build anything from the basic backyard fence to an ornate, double sided, wood capped, rock pillar fence and anything in between.
All of our attachments are done with screws, bolts, or clamps. We don't use any nails or staples at all.
We take pride in our fences and we stand behind them.


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A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins from hypo- to hyperphosphorylated species, events that are highly dependent on casein kinase 1ε (termed DOUBLETIME [DBT] in Drosophila melanogaster) and necessary for normal clock progression. Drosophila PER (dPER) functions in the negative limb of the clockworks by presumably binding to the transcription factor CLOCK (CLK) and inhibiting its transactivation activity. Here, we identify a small region on dPER that is conserved with mammalian PERs and contains the major in vivo DBT binding domain, termed dPDBD (for dPER DBT binding domain). This domain is required for the manifestation of molecular and behavioral rhythms in vivo. In the absence of the dPDBD, the dPER protein is present at constant high levels throughout a daily cycle, undergoes little phosphorylation, and is severely impaired in its ability to function as a transcriptional repressor. Our findings indicate that the binding of dPER to CLK is not sufficient for transcriptional inhibition, implicating a more indirect mode of action whereby dPER acts as a molecular bridge to "deliver" DBT and/or other factors that directly repress CLK-dependent gene expression.
Circadian (≅24-h) rhythms are a pervasive feature of the temporal organization exhibited by life-forms ranging from bacteria to plants and humans (30). These rhythms are driven by intracellular clocks or pacemakers that share a similar mechanism. The central organizing principle is based on interconnected transcriptional-translational feedback loops that produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time, and transducing temporal signals to downstream effector pathways (17, 72). Work using Drosophila melanogaster has been instrumental in our understanding of clock mechanisms in general and especially in animals, since analogous clock genes operate in both systems (18, 29).
The intracellular clock mechanism in Drosophila is largely depicted as two interconnected transcriptional feedback loops with overlaying posttranslational regulatory circuits (4, 28). Prominent players in the first or "major" loop are PERIOD (PER; herein referred to as Drosophila PER [dPER]), TIMELESS (TIM), CLOCK (dCLK) and CYCLE (CYC). dCLK and CYC are transcription factors of the basic helix-loop-helix/PAS (Per-Arnt-Sim) superfamily that heterodimerize to stimulate the daily transcription of dper and tim, in addition to other clock and downstream genes (3, 15, 56). A key aspect of the temporal regulation in dCLK-CYC activity involves the phase-specific accumulation of dPER and TIM in the nucleus, whereby dPER (and less likely TIM) interacts with the dCLK-CYC heterodimer, inhibiting transactivation (15, 40, 41, 55). After several hours in the nucleus, the levels of TIM and dPER undergo sharp decreases, relieving autoinhibition and initiating another round of dCLK-CYC-dependent transcription. In the so-called "second" loop, cycles in dClk expression are driven by the alternating actions of two bZip transcription factors, VRILLE and PDP1ε (13, 25). A very similar circuitry operates in mammalian circadian clocks, which includes the participation of mammalian CLK (mCLK), BMAL1 (homolog of CYC), and several PER homologs (mammalian PER protein 1 [mPER1], mPER2, and mPER3) (37).
Temporal changes in dPER phosphorylation are thought to play a central role in controlling dPER function such that it operates in a phase-specific manner to inhibit dCLK-CYC-dependent transcription only during certain times in a daily cycle (i.e., from approximately early/mid-night to midday) (4). Newly synthesized dPER is initially present as a hypophosphorylated variant(s) in the late day/early night, progressively increasing in the extent of phosphorylation such that by the late night/early day only hyperphosphorylated species are detected (19). Phosphorylation has been linked to regulation of dPER stability (26, 35, 38, 53), nucleocytoplasmic distribution (2, 7, 14, 44, 45, 54), and possibly transcriptional repressor potency (51).
The DOUBLETIME (DBT) kinase (homolog of mammalian casein kinase 1ε [CK1ε]) is a key kinase controlling the temporal program underlying dPER phosphorylation and stability (35, 53). In a presumptive dbt-null mutant (dbtP), dPER is hypophosphorylated and attains high constant levels (53). DBT associates with dPER through most or all of its daily life cycle (36) and is thought to modulate the phosphorylation and stability of dPER in both the cytoplasm and the nucleus (53). Newly synthesized dPER in the cytoplasm is rapidly degraded in a DBT-dependent manner. As the levels of TIM increase during the day, it binds dPER (but see reference 48) and somehow protects it against DBT-mediated degradation. The interaction of dPER with TIM also stimulates but is not obligatory for nuclear localization of both proteins (49, 57, 60, 68). Nuclear entry/accumulation of dPER and TIM are also controlled by at least three kinases, DBT, CK2, and SHAGGY/glycogen synthase kinase 3β, with the former two likely directly targeting dPER and the last likely targeting TIM (2, 7, 14, 44, 45, 47). In the nucleus, DBT and TIM continue to modulate the metabolism of dPER, whereby the earlier drop in the abundance of TIM somehow accelerates the progressive multiphosphorylation of dPER (36, 55). Hyperphosphorylated isoforms of dPER are targeted to the 26S proteasome by the F-box protein SLIMB (26, 38). Rapidly declining levels of dPER severely diminish or terminate its repressor function on dCLK-CYC activity. In addition to kinases, protein phosphatase 2A and possibly other phosphatases regulate the phosphorylated state of dPER and modulate the clockworks (58).
mPERs undergo similar cycles in phosphorylation, abundance, and subcellular localization, with prominent roles for CK1ε and its very close variant CK1δ (20, 42). Indeed, mutations that affect the phosphorylation of human PER2 (65) or the activity of CK1δ (71) are causally linked to familial sleep disorders. Moreover, it appears that the cyclical phosphorylation of one or more clock proteins is the core biochemical foundation in generating an oscillator with circadian properties (50). Here we identify the major or sole region on dPER that mediates stable interactions with DBT. In the absence of this region (herein termed dPER DBT binding domain [dPDBD]), the altered dPER protein [dPER(Δ)] does not support molecular or behavioral rhythms and is constitutively expressed at elevated levels in a hypophosphorylated manner. Surprisingly, although dPER(Δ) retains its ability to bind TIM and dCLK it is a very poor repressor, indicating that the stable association of dPER with dCLK is not sufficient to block transcription. Our findings offer new perspectives on the role of dPER in the negative limb of the Drosophila clockworks.
Plasmids.The pAct-dper, pMT-dClk-V5, and pMT-dbt-V5 plasmids were described previously (9, 34, 38). To construct either full-length or truncated versions of dper-V5, the desired coding regions of dper were amplified by PCR in the presence of pAct-dper and the relevant fragments subcloned into pAc5.1/V5-His (Invitrogen). The pAct-dper(Δ755-809)-V5 construct was generated by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) and the internally deleted dper fragment subcloned into the pAc5.1/V5-His vector. A similar strategy was used to generate the pAct-dper(S/T9A) plasmid, which encodes a nontagged dPER variant that has nine conserved Ser/Thr residues in the region between amino acids (aa) 755 and 809 changed to Ala residues. All the dper constructs were verified by DNA sequencing. To generate pAct-tim-3HA, the entire tim open reading frame was amplified by PCR in the presence of pAct-tim (9) and exchanged for the slimb fragment in the pAct-slimb-3HA vector (38).
Transgenic flies.To generate transgenic flies that produce the dPER(Δ) protein, we used a previously described CaSpeR-4-based transformation vector containing a 13.2-kb genomic dper insert that was modified with sequences encoding the hemagglutinin (HA) epitope tag and a stretch of histidine residues just upstream of the dper translation stop signal, termed 13.2(per+-HA10His) (herein referred to as per+-HAHis) (40). Deletion of sequences encoding aa 755 to 809 from dPER was performed using a QuikChange site-directed mutagenesis kit (Stratagene, CA) with an appropriate dper genomic subfragment, confirmed by DNA sequencing, and reconstructed into the above-mentioned transformation vector to yield 13.2(per(Δ)-HA10His) [herein referred to more simply as per(Δ)-HAHis]. Transgenic flies were generated by Genetics Services Inc. (Sudbury, MA) using standard P element-mediated transformation techniques and w1118 embryos as hosts. Three independent germ line transformants bearing the per(Δ)-HAHis transgene in a per+ background (w1118per+; herein referred to as wper+) were obtained and then crossed into a wper01 genetic background to yield wper01; per(Δ)-HAHis. Similar procedures were also performed with the original version of the per+-HAHis transgene (40) to generate appropriate control groups expressing a wild-type version of the recombinant dPER protein in the same w1118 and wper01 genetic backgrounds. For the wild-type control transgenic flies, we show behavioral and molecular results obtained with one of the lines (M16), which are representative of other independent lines bearing the per+-HAHis transgene (data not shown).
Locomotor activity.The locomotor activities of individual flies were measured as previously described using the monitoring system from Trikinetics (Waltham, MA) (27). Young adult male flies were used for the analysis and kept in incubators at 25°C, exposed to at least 4 days of 12 h of light followed by 12 h of dark (12:12LD, where zeitgeber time zero [ZT0] is defined as the time when the light phase begins) and subsequently kept in constant-darkness conditions (DD) for 5 to 8 days. The locomotor activity data for each individual fly were analyzed using the FaasX software (Fly Activity Analysis Suite for MacOSX), which was generously provided by F. Rouyer. Periods were calculated for each individual fly using chi-square periodogram analysis and pooled to obtain a group average for each independent transgenic line or genotype. Power is a quantification of the relative strength of the rhythm during DD. Individual flies with a power of ≥10 and a "width" value (denoting the number of peaks in 30-min increments above the periodogram 95% confidence line) of 2 or more were considered rhythmic.
Methods using cultured S2 cells.S2 cells were obtained from Invitrogen and transfected using Cellfectin reagent (Invitrogen) as described previously (38). Exogenous DBT was induced in cells transfected with pMT-dbt by adding CuSO4 to a final concentration of 500 μM in the media. RNA-mediated interference (RNAi) directed against endogenously expressed slimb was performed as described previously (38, 70). Briefly, after the addition of double-stranded RNA against slimb (38), cells were allowed to recover for 2 days, transfected with the desired plasmids, and incubated for a further 1.5 days before being harvested. dCLK-dependent transactivation using a luciferase (luc) reporter assay was performed as described previously, with slight modifications (15). Briefly, S2 cells were placed in 12-well plates and transfected with pAct-dper(1-1224) and/or pAct-dper(Δ) plasmids at the indicated amounts, along with 10 ng of perEluc, 30 ng of pAct-β-gal-V5/His, and 2 ng of pMT-dClk-V5. One day after transfection, dClk expression was induced with 500 μM CuSO4 (final in the media), and after another day cells were washed in phosphate-buffered saline, followed by lysis in 300 μl of reporter lysis buffer (Promega). Aliquots of cell extracts were assayed for β-galactosidase (β-Gal) and luciferase activities using the luciferase assay system and protocols supplied by the manufacturer (Promega).
Immunoblotting.To prepare cell extracts for immunoblotting of proteins in cultured S2 cells, the cells were washed with phosphate-buffered saline and treated as previously described (34, 38), except that additional buffers were used depending on the protein sought (extracts prepared for immunoprecipitation are described below). When dClk plasmids were not included in the transfection, cells were homogenized in our standard EB1 solution (10 mM HEPES [pH 7.5], 5 mM Tris-HCl [pH 7.5], 50 mM KCl, 10% glycerol, 2.5 mM EDTA, 5 mM dithiothreitol [DTT], 0.2% Triton X-100, and 2.5 mM NaF), with the addition of a protease inhibitor cocktail at the manufacturer's recommended concentration (Roche) (34, 38). When we wanted to detect recombinant dCLK, extracts were prepared in harsher conditions with radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 0.1% sodium dodecyl sulfate [SDS]), with the addition of a protease inhibitor cocktail (Roche). Prior work showed that harsher conditions are more efficient for extracting dCLK (references 34 and 73 and data not shown). In the case of fly material, flies were collected by freezing at the indicated times in LD and total fly head extracts prepared using either mild (EB1) or harsh (RIPA) conditions, as indicated. Gels with differing polyacrylamide concentrations were used to resolve different proteins (6% for dPER, TIM, and dCLK, and 12% for DBT). In the case of dCLK, 5% Criterion gels (Bio-Rad) were also used. Primary antibodies were used at the following dilutions: for anti-V5 (Invitrogen), 1:5,000; for anti-PER (GP73), 1:3,000 (62); for anti-HA (12CA5; Roche), 1:2,000; for anti-HA (3F10; Roche), 1:1,000; for anti-TIM (TR3), 1:2,000 (62); for anti-dCLK (GP208 [34] [see below] or GP-47 [32]), 1:2000; and for anti-DBT (GP292), 1:2,000 (see below).
To generate antibodies to DBT, we used PCR to amplify two dbt cDNA sequences that encode either aa 1 to 177 (dbt-N) or aa 253 to 440 (dbt-C) and cloned the relevant fragments into pET-21b (Novagen). The oligonucleotide primers used in the PCR were as follows (dbt sequences are underlined): 5′-ATCAATTCGATGGAGCTGCGCGTGGG-3′ and 5′-ATAAGCTTGGCAGTGCCCGTGAGGTTC-3′ for dbt-N, and 5′-ATGAATTCGAACTTCTGTCGCCAGATG-3′ and 5′-ATAAGCTTTTTGGCGTTCCCCACGCC-3′ for dbt-C. Purified fusion proteins were used to produce antibodies in guinea pigs (Cocalico Biologicals, Reamstown, PA). The most specific antibodies to DBT were obtained when using DBT-N as the immunogen, and one antiserum (GP292) was used in this study. We also raised novel anti-dCLK antiserum in guinea pigs (Cocalico Biologicals, Reamstown, PA) by use of the same immunogen previously described (40), and in this study we used GP208, which showed the highest dCLK staining intensity with low background (data not shown). Relevant bands on films were quantified using a charge-coupled-device camera and Alphaimager 2200 V5.5 software from Alpha Inotech Corporation.
Immunoprecipitation.For immunoprecipitations, cell extracts, either from S2 cells or fly heads, were prepared using modified RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) with the addition of a protease inhibitor cocktail (Roche). To the extracts, 3 μl of anti-HA (12CA5), anti-V5, or anti-PER (GP73) antibody was added, depending on the target protein sought, and incubated with gentle rotation for 3 to 5 h at 4°C followed by the addition of 5 μl of Dynabeads Protein A (Invitrogen) with a further incubation of 1 to 2 h. Beads were collected using DynalMPC. Immune complexes were mixed with 30 μl of 1× SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and incubated for 10 min at 65°C, and the resulting supernatants were revolved by immunoblotting as described above. To detect interactions between DBT and dPER in flies (see Fig. 6A), 20 μl of either HA-agarose (Sigma) or the nonspecific V5-agarose (Sigma) was added to total fly head extracts. Immune complexes were mixed with 0.1 M glycine-HCl (pH 2.7) buffer and incubated for 10 min at 25°C to release bound proteins. Beads were collected by light centrifugation and the resulting supernatants mixed with 4× SDS sample buffer and resolved on 12% polyacrylamide gels, followed by immunoblotting in the presence of anti-DBT antibodies. Phosphatase treatment of immune complexes was performed as described previously (40) except that Dynabeads Protein A was used instead of Gammabind Plus (Pharmacia).
In vitro phosphorylation.For the in vitro kinase assay, pAct-dper-V5 or pAct-dper(Δ)-V5 was transfected into S2 cells and incubated for 48 h (see Fig. 2C). Extracts were prepared in EB1 buffer (see above), and dPER or dPER(Δ) was immunoprecipitated using anti-dPER antibodies (GP73) and Gammabind Plus Sepharose beads (Pharmacia), as described above. Subsequently, the immune complexes were equilibrated in CK1 reaction buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 5 mM dithiothreitol, and 200 μM ATP) and subjected to an in vitro kinase reaction with purified recombinant CK1δ (New England Biolabs, Beverly, MA) according to the supplier's protocols. Briefly, 500 units of recombinant CK1δ and 10 μCi of [γ-32P]ATP were added to beads containing either dPER or dPER(Δ) in 50 μl of CK1 reaction buffer on ice, followed by incubation for 30 min at 30°C. Reactions were terminated by the addition of 2× SDS-PAGE sample buffer and resolved on 6% polyacrylamide gels. Subsequently, gels were dried and radioactive bands visualized using a Typhoon 9400 Imager, and the intensity was quantified using Imagequant software (Molecular Dynamics).
RT-PCR.The relative mRNA levels of dper and dClk were measured by semiquantitative reverse transcription-PCR (RT-PCR) essentially as described previously (46). Briefly, 2 μg of total RNA was used to synthesize cDNA, and a 2-μl aliquot of the reaction was further processed by PCR in a final volume of 50 μl using either dper- or dClk-specific primers. For dper amplification, primers were P7197 (5′-TCTACATTATCCTCGGCTTGC-3′) and P6869 (5′-TAGTAGCCACACCCGCAGT-3′), as previously described (46). For dClk, we used the following set of primers: dClk6856F (5′-TCCTGAAGTCCACGATAGCC-3′) and dClk7105R (5′-TTCCGAGGCGTAAAAGATGG-3′). To control for sample-to-sample differences in total RNA, we also included primers for the noncycling mRNA coding for CBP20 (cap binding protein 20) by use of the primers CBP495R (5′-CAACAGTTTGCCATAACCCC-3′) and CBP362F (5′-GTCTGATTCGTGTGGACTGG-3′), as previously described (46). PCR products were separated and visualized by electrophoresis on 2% agarose gels containing Gelstar (Cambrex Co.), and the bands were quantified using a Typhoon 9400 Imager.
ChIP assays.Chromatin immunoprecipitation (ChIP) assays were performed as described previously (73) with the following modifications. Adult flies were frozen at the indicated times in LD, and isolated heads (<900 μl) were gently homogenized for 10 min at 25°C in <5 volumes of XIP homogenization buffer (50 mM HEPES at pH 8.0, 140 mM NaCl, 0.4% Igpel CA-630, 0.2% Triton X-100, 1% HCHO, 1 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM Na3VO4, and 1 mM NaF). Glycine was then added to a final concentration at 0.125 M to stop the cross-linking reaction. Homogenates were filtered with 100-μm nylon meshes to remove cuticle. The cross-linked nuclei were harvested by centrifugation at 800 × g for 5 min, washed three times with XN wash buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM Na3VO4, and 1 mM NaF), and stored at −80°C for future use. Extracts used for ChIP were prepared by sonicating the cross-linked nuclei in sonication buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.4% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM Na3VO4, and 1 mM NaF) 15 times for 10 s each (150 s in total) by use of a Misonix XL2000 sonicator set at 3. The average size of sheared DNA fragments was <500 bp on agarose gels (data not shown). After centrifugation at 25,000 × g for 10 min, the supernatants were quantified by Bradford assay (7a). Fifty micrograms of extract was set aside as input, and 500 μg of extract was precleared and subjected to immunoprecipitation using GP-47 (dCLK antisera made in guinea pig [32]) or normal guinea pig serum (GPS). The preclearance, immunoprecipitation, and DNA extraction were performed as described previously (73). DNA from immunoprecipitates and inputs was suspended in 50 μl of 1× TE. This suspension was subjected to "hot" PCR after being diluted 1:20 for immunoprecipitated samples and 1:300 for input DNA (thus equaling 0.33% of input). For each 20 μl of PCR mixture, 1 μl of diluted DNA was used as the template. Semiquantitative "hot" PCR was performed as described previously (73). Bands from PCR products were resolved on polyacrylamide gels and quantified by densitometry. For each time point and genotype, the background signal from material collected in the presence of GPS antiserum was subtracted from the values obtained using anti-dCLK antibodies. Finally, dCLK-specific values were converted to percentages of input by use of the following formula: % input = (dCLK signal/10 × 20)/(input signal × 300) × 100%.
A region between aa 755 and 809 is essential for DBT-dependent hyperphosphorylation and SLIMB-mediated degradation of dPER in S2 cells.Prior work using dPER fragments beginning at the amino terminus and fused to β-Gal implicated a stretch of 231 residues between aa 638 and 876 in dPER as necessary for its hyperphosphorylation and temporal instability in flies (16) (Fig. 1A). We previously showed that the DBT-dependent progressive phosphorylation and subsequent SLIMB-mediated degradation of dPER can be recapitulated in a simplified cell culture system by expressing recombinant proteins in Drosophila Schneider (S2) cells (38). In this system, dPER is expressed from a constitutive promoter (pAct), whereas exogenous DBT is induced using the copper-inducible metallothionein promoter (pMT). To further delineate the region on the 231-aa stretch of dPER presumably mediating DBT-dependent hyperphosphorylation and enhanced degradation, we generated additional versions of dPER (fused to the V5 epitope to facilitate detection) with truncations between aa 638 and 876 and evaluated their responses following DBT induction in cultured S2 cells (Fig. 1B).
As previously shown using this experimental paradigm, the induction of DBT evokes dramatic increases in the phosphorylation of full-length dPER [dPER(1-1224)] that are readily observed as temporal decreases in electrophoretic mobility (38) (Fig. 1B, lanes 1 to 3, and 2A, lanes 1 to 4). Under the conditions used, full-length dPER exhibits a highly smeared pattern after 24 h of inducing dbt, which upon further incubation progresses to exclusively hyperphosphorylated species that are less stable (e.g., Fig. 2A, compare lanes 1 and 4). Notably different from this pattern, dPER(1-638), dPER(1-697), and dPER(1-754) fragments show only narrow mobility shifts that are characterized by the appearance of one or two sharp bands following induction of dbt, most likely due to limited phosphorylation (Fig. 1B, lanes 4 to 12). Although the mobility shifts do not appear as extensive for dPER(1-876) as for dPER(1-1224), this fragment displays a more smeared mobility pattern indicative of multiphosphorylation (lanes 13 to 15), consistent with prior work with transgenic flies expressing the dPER(1-876)-β-Gal fusion (Fig. 1A) (16). We also analyzed an extensive series of other dPER fragments and observed that strong DBT-dependent hyperphosphorylation requires aa 638 to 876 (reference 10 and data not shown). Together, our results suggest that although there are likely to be multiple areas on dPER that are phosphorylated or contribute to phosphorylation, the region between aa 755 and 876 plays a major role in the DBT-dependent hyperphosphorylation of dPER (Fig. 1B).
Based on several phosphorylation site prediction programs, there are multiple plausible CK1ε sites clustered between aa 755 and 809 of dPER (data not shown). We thus generated a dPER variant internally deleted from aa 755 to 809 [referred to as dPER(Δ)] and probed its response to DBT induction. Without induction of exogenous dbt, both the wild-type dPER(1-1224) and dPER(Δ) versions are exclusively hypophosphorylated and produced at comparable levels (Fig. 2A, compare lanes 1 and 5). Following dbt induction, the progressive accumulation of highly phosphorylated dPER was markedly delayed or attenuated for dPER(Δ) compared to that for full-length dPER (Fig. 2A). Importantly, the overall levels of dPER(Δ) did not undergo significant changes even after 36 h following induction of DBT, in sharp contrast to the wild-type control protein (Fig. 2A, compare lane 4 to lane 1 and lane 8 to lane 5). These results indicate that the region stimulating DBT-dependent hyperphosphorylation is also necessary for enhanced degradation, consistent with the observation that hyperphosphorylation of dPER is accompanied by rapid decreases in levels (26, 35, 38, 53). Similar results using a slightly smaller internal deletion (Δ768-792, according to the numbering used in this study) were recently obtained by others (51a). Moreover, while the degradation of dPER is significantly attenuated by RNAi targeting endogenous slimb expression as previously shown (38) (Fig. 2B, compare lanes 4 and 5), there was no effect on the levels or phosphorylated status of dPER(Δ) under the same conditions (Fig. 2B, compare lanes 2 and 3). This indicates that our inability to detect highly phosphorylated isoforms of dPER(Δ) is not due to their rapid degradation but most likely reflects inefficient phosphorylation (Fig. 2B). Indeed, unlike full-length dPER purified from S2 cells, which is extensively phosphorylated in vitro by recombinant mammalian CK1ε/δ (purified DBT does not exhibit in vitro kinase activity for unknown reasons, as previously noted [e.g., reference 63]), dPER(Δ) is a very poor substrate (Fig. 2C, compare lanes 3 and 4).
We considered the possibility that the region between aa 755 and 809 contains multiple phosphorylation sites that underlie the DBT-mediated hyperphosphorylation of dPER. There are 12 Ser/Thr residues in this region, of which 9 are highly conserved in dPER from other drosophilids (data not shown). We therefore altered all the conserved nine Ser/Thr residues to Ala, generating dPER(S/T9A) (Fig. 2D). Surprisingly, dPER(S/T9A) behaves similar to wild-type dPER in terms of phosphorylation and temporal changes in abundance when coexpressed with DBT. These findings suggest an alternative explanation whereby aa 755 to 809 are important for DBT binding and that this association subsequently facilitates DBT-mediated hyperphosphorylation of dPER at other sites. Indeed, the appearance of a minor fraction of hyperphosphorylated isoforms of dPER(Δ) in some experiments (e.g., that shown in Fig. 2A) further suggests that the region between aa 755 and 809 is not a major site directly modified by multiple phosphorylation events [although we cannot rule out the possibility that the DBT-induced multiphosphorylated species of dPER(Δ) arise from aberrant phosphorylation events].
The region encompassing dPER aa 755 to 809 is a major binding domain for DBT.To test if dPER aa 755 to 809 play a role in the association of DBT with dPER, we performed immunoprecipitation assays between recombinant dPER(1-1224) or dPER(Δ) and DBT proteins expressed in S2 cells. Under the experimental conditions used, about two- to threefold less dPER(Δ) copurifies with DBT, even though there is more dPER(Δ) present in the extract than there is dPER(1-1224) (Fig. 3A, lanes 2 and 3; results from two independent experiments are shown). We varied the relative and absolute concentrations of the dbt and dper plasmids during transfection and still obtained very similar results, whereby less dPER(Δ) than dPER(1-1224) copurifies with DBT (data not shown). The observation that the interaction between dPER(Δ) and DBT is severely decreased but not abolished is consistent with the low levels of hyperphosphorylation (Fig. 2A, lanes 5 to 8). Importantly, dPER(Δ) stably interacts with TIM or dCLK with a relative efficiency the same as or better than that of the full-length dPER control (Fig. 3B, lanes 2 and 3, and C, lanes 2 and 4). These results show that deletion of aa 755 to 809 in dPER specifically affects its ability to stably interact with DBT. Similar results were obtained when we used GST-DBT bound to resins to examine the interaction with dPER and dPER(Δ) (data not shown). The fact that dPER(Δ) strongly binds TIM and dCLK indicates that the inability to associate with DBT is not due to nonspecific issues such as gross misfolding of the internally deleted dPER variant. Based on these and other results shown below we refer to the region between aa 755 and 809 as the dPDBD (for dPER DBT binding domain).
dPER(Δ) is highly impaired in its ability to inhibit dCLK-dependent transcriptional activation.We next sought to evaluate the ability of dPER(Δ) to inhibit dCLK-CYC-mediated transactivation of gene expression using the well-established assay developed with S2 cells (15). In this system, dCLK is exogenously expressed (the levels of endogenously produced CYC are sufficient) and stimulates expression of a luc reporter fused to an E-box containing circadian promoter (usually dper or tim). Recent work has shown that dPER alone (i.e., without coexpression of TIM) acts as a potent repressor of dCLK-dependent transactivation in the cultured S2 cell system (10, 51, 69), consistent with findings with flies (14, 55). Unlike full-length dPER, dPER(Δ) has little repressor effect, exhibiting at most a 20% reduction in reporter gene expression at the highest dose tested (Fig. 4A). Interestingly, the dPDBD falls within a much larger region of dPER (aa 764 to 1034), previously identified as the minimal region giving over 50% inhibition of dCLK-dependent transcription in S2 cells (termed CCID [for CLK-CYC-inhibitory domain]) (10) (Fig. 1A). Variations in the total levels of dPER(1-1224) and dPER(Δ) cannot account for the differences in repressor activity (e.g., Fig. 2A, compare lanes 1 and 5). Furthermore, in our system we did not observe significant differences in the subcellular distributions of dPER(Δ) and to dPER(1-1224) (data not shown and Discussion). The levels of recombinant dCLK in cells coexpressing either dPER(1-1224) or dPER(Δ) (Fig. 4B) are comparable, indicating that the severely attenuated repressor function of dPER(Δ) is not due to secondary effects on dCLK metabolism (for reasons that are still unclear, the levels of dCLK slightly increase with high amounts of dPER [e.g., Fig. 4B, compare lane 1 to 4 and 7]). Finally, although we cannot rule out subtle effects on dCLK phosphorylation, dPER(1-1224) inhibits dCLK activity without any gross increases in dCLK phosphorylation. These results suggest that in our cell culture system, the hyperphosphorylation of dCLK is not absolutely required for its repression by dPER, consistent with our earlier in vitro results (41).
Thus, the dPDBD is critical for the ability of dPER to act as an efficient repressor of dCLK-CYC (similar results were recently obtained by Nawathean and colleagues [51a]). We note that dPER(Δ) exhibits highly impaired repressor activity even in the absence of dbt induction, where only hypophosphorylated isoforms of dPER(Δ) and wild-type dPER are detected (e.g., Fig. 2A, compare lanes 1 and 5), suggesting that the lack of extensive phosphorylation or hyperphosphorylation does not explain the inability of dPER(Δ) to block dCLK-dependent transcription (see Discussion). Moreover, because dPER(Δ) interacts with dCLK (Fig. 3; also see Fig. 6), it is clear that the binding of dPER to dCLK is not sufficient for transcriptional inhibition. Nonetheless, the ability of dPER(Δ) to bind dCLK might explain why coexpression of dPER(Δ) interferes with the repressor activity of wild-type dPER (Fig. 4C and Table 1; also see below), suggesting that dPER(Δ) acts in a dominant-negative manner with regards to transcriptional autoinhibition.
Arrhythmic behavior in dper(Δ)-expressing flies.To evaluate the physiological significance of the dPDBD in clock function, we generated transgenic flies harboring a dper transgene internally deleted for sequences encoding aa 755 to 809 of dper. The internal deletion was engineered into a previously characterized vector that contains a 13.2-kb dper genomic fragment [13.2(per+)] and also includes a HA epitope tag and a stretch of His moieties (10His) at the carboxy terminus of the dper open reading frame (per+-HAHis) protein, facilitating purification and surveillance of the recombinant protein (40). Several independent lines of transgenic flies bearing the dper(Δ) transgene [13.2(per(Δ)-HA10His)] were obtained and evaluated for circadian functionality in the per-null wper01 (39) genetic background [wper01; 13.2(per(Δ)-HA10His), herein more simply referred to as wper01; per(Δ)-HAHis]. Prior work showed that transgenic per01 flies harboring 13.2(per+)-based transgenes manifest robust locomotor activity rhythms with periods similar to those of per+ wild-type controls (Table 1).
For behavioral analysis, flies were initially entrained under standard conditions of 12:12LD, where ZT0 is defined as the time when the light phase begins, at 25°C, followed by at least 1 week in DD. In contrast to the wper01; per+-HAHis control transgenic flies, all the independent dper(Δ)-containing transformants were arrhythmic (Table 1 and data not shown). When dper(Δ) was introduced into a per+ background [w; per(Δ)-HAHis)], the proportion of arrhythmic flies significantly increased, and for those displaying rhythmic activity the amplitudes were strongly reduced and the periods lengthened by 3 to 4 h (Table 1). The semidominant effects of dPER(Δ) on behavioral rhythms might arise from an obstruction of the ability of wild-type dPER to inhibit dCLK-dependent transcription, as observed for S2 cells (Fig. 4C).
High constant levels of exclusively hypophosphorylated dPER(Δ) in flies.We next investigated the temporal regulation of dPER phosphorylation and abundance by analyzing head extracts prepared from flies collected at different times of day. The levels and time-of-day-specific phosphorylation of the control dPER-HAHis protein in wper01; per+-HAHis transgenic flies undergo daily cycles that are indistinguishable from those for endogenously expressed dPER (Fig. 5A, top panel, lanes 1 to 6, and data not shown). Moreover, TIM also exhibits normal cycles in abundance (Fig. 5A, bottom panel, lanes 1 to 6). Consistent with the behavior results, dPER(Δ)-HAHis does not undergo noticeable changes in mobility or abundance throughout a daily cycle (Fig. 5A, top panel, compare lanes 9 to 14 to lanes 7 and 8). Also, the overall abundance of dPER(Δ)-HAHis is approximately two- to threefold higher than the daily peak values for the wild-type control dPER protein (Fig. 5A, top right panel, compare lanes 8 and 9; also see Fig. 6B and D). In agreement with prior work (19), λ phosphatase treatment revealed that the electrophoretic mobility variants of dPER-HAHis are due to differential phosphorylation (Fig. 5B, lanes 1 to 3). In sharp contrast, dPER(Δ)-HAHis manifests only a slight increase in mobility when treated with λ phosphatase, indicating that the overwhelming majority is hypophosphorylated with some limited phosphorylation (Fig. 5B, compare lanes 4 and 5). Unlike the situation for S2 cells, for which we occasionally detected low levels of highly phosphorylated dPER(Δ) following dbt induction (Fig. 2A), we never observed extensively phosphorylated dPER(Δ) produced in flies, even when films were exposed for long periods (data not shown). It is possible that low-efficiency hyperphosphorylation of dPER(Δ) is possible in S2 cells where the relevant target proteins are highly expressed. TIM in wper01; per(Δ)-HAHis flies displayed low but constant levels during the day and higher constant amounts during the night (Fig. 5A, bottom panel, lanes 9 to 14). This profile is similar to that observed for per0 flies, where circadian cycling of TIM is abolished but daily changes arise from the direct photosensitivity of TIM (49, 74).
We recently showed that when head extracts are prepared using harsh conditions, immunoblotting analysis of dCLK reveals that its levels are relatively constant throughout a daily cycle (Fig. 5C, lanes 1 to 7) (34, 73). Moreover, there are several different dCLK phosphoisoforms (non- or hypophosphorylated to intermediately phosphorylated and hyperphosphorylated). The hyperphosphorylated isoform(s) is detected only in the late night/early day, is absent from per0 flies, and likely requires DBT activity (34, 73). This profile differs from that found in our earlier work, in which we used mild extraction conditions and showed a biochemical cycle in dCLK abundance with trough amounts reached during the late day and peak values attained in the late night/early day (e.g., Fig. 5C, compare lanes 8 and 9) (40). In contrast to harsh conditions, mild treatments do not efficiently release dCLK from chromatin, and it is extracted mainly during times of dPER-mediated repression (mid-night to midday) when dCLK is hyperphosphorylated and displaced from chromatin (73).
To examine the status of dCLK in dPER(Δ)-expressing flies, we collected flies at times in a daily cycle that represent relatively active (e.g., ZT11.5 and 14) and inactive (e.g., ZT23.5 and 2) phases of dCLK-CYC-driven circadian transcription, respectively. When we used harsh extraction conditions, the overall levels of dCLK were comparable in both wper01; per+-HAHis and wper01; per(Δ)-HAHis flies (Fig. 5C, lanes 3, 4, 6, and 7). A notable difference, however, is that hyperphosphorylated dCLK does not accumulate in wper01; per(Δ)-HAHis flies (Fig. 5C, compare lanes 3 and 4; to simplify, we designated all the nonhyperphosphorylated dCLK as hypophosphorylated). These results are consistent with the suggestion that dPER acts as a bridge to enhance dCLK phosphorylation by DBT (73), a function impaired in the dPER(Δ) mutant (Fig. 6A and Discussion). As previously shown (5, 40), with mild extraction conditions dCLK is readily observed in extracts prepared from flies collected at ZT23.5 but not in those from ZT11.5 (Fig. 5C, compare lanes 8 and 9). However, irrespective of daily time, dCLK is not observed in wper01; per(Δ)-HAHis head extracts prepared using mild extraction procedures (Fig. 5C, lanes 10 and 11), reinforcing the contention that dCLK is constitutively bound to chromatin in the presence of dPER(Δ) (Fig. 7).
dPER(Δ) interacts with dCLK and TIM but not DBT in flies.To examine the in vivo interaction of dPER(Δ) with DBT, we collected wper01; per(Δ)-HAHis and wper01; per+-HAHis flies at ZT2 and 14, times in a daily cycle when wild-type dPER is present mainly as either hyper- or hypophosphorylated isoforms, respectively (Fig. 6B, top panel, compare lanes 1 and 3). Wild-type dPER stably interacts with DBT throughout a daily cycle (Fig. 6A, lanes 2 and 6) as previously reported (36) (the slightly stronger DBT staining at ZT2 is likely due to the higher overall levels of dPER at this time [Fig. 6B, top panel, compare lanes 1 and 3]). No DBT signal was observed when an irrelevant antibody was used (e.g., Fig. 6A, compare lanes 1 and 2), demonstrating specificity. In sharp contrast, dPER(Δ) does not copurify with DBT (Fig. 6A, compare lane 3 to lane 2 and lane 6 to lane 7), despite the much higher overall levels of dPER(Δ) than of wild-type dPER (Fig. 6B, top panel). Gross differences in DBT levels cannot explain the lack of interaction between dPER(Δ) and DBT (Fig. 6B, bottom panel). As with the difference between dPER(Δ) in flies and that in S2 cells, with more-dramatic effects on hyperphosphorylation seen for the former (compare Fig. 2A and 5A), the interaction between dPER(Δ) and DBT appears completely abolished in flies, unlike the situation for the cultured cells. As noted above, this could simply be a function of the higher concentrations attained in S2 cells or of other in vivo constraints that are not present in S2 cells. For example, the in vivo interaction between dPER and DBT might be stimulated by other factors that directly or indirectly interact with the dPDBD. In any event, our results strongly suggest that the stable interaction of dPER with DBT is required for dPER hyperphosphorylation and that the dPDBD is the major or sole region mediating this association in vivo.
Consistent with the S2 cell results, both dPER and dPER(Δ) bind dCLK and TIM (Fig. 6C, lanes 2 and 3). The higher abundance of TIM copurifying with dPER(Δ) compared to that copurifying with dPER (Fig. 6C, bottom panel, lanes 2 and 3) is likely a result of the increased levels of dPER(Δ) in the extract (Fig. 6B, top panel, compare lanes 1 and 2). In the case of dCLK, equal amounts are copurified with dPER and dPER(Δ) (Fig. 6C, top panel), most likely because dCLK levels are limiting (5). Furthermore, because at ZT23.5 dPER is highly phosphorylated compared to dPER(Δ) (e.g., Fig. 6D, top panel, compare lanes 2 and 3), our results suggest that the phosphorylated state of dPER has little if any effect on its ability to bind dCLK.
dPER(Δ) is inefficient in the negative limb of the transcriptional feedback circuitry.In wper01; per+-HAHis transgenic flies, dper RNA attains peak abundance at ∼ZT12 and trough levels by ∼ZT0 (Fig. 7A), similar to the endogenous dper temporal profile. Consistent with the luc-based results obtained with S2 cells (Fig. 4), the average daily levels of dper RNA in wper01; per(Δ)-HAHis flies are higher than in the control wper01; per+-HAHis situation (Fig. 7A). Both genotypes show similar peak values for dper transcript levels, suggesting that in the presence of dPER(Δ) the dper negative feedback loop is essentially locked in the "derepressed" mode. Interestingly, during the night phase dper(Δ) transcript levels exhibit a slight but reproducible decline (Fig. 7A). Although the reason for this night-specific decline is not clear, evidence indicates that at least under some circumstances, TIM and/or CRY can function as repressors of dCLK-CYC activity (11, 15, 41). Presumably, the light-induced degradation of TIM and/or CRY would reduce its capacity to inhibit dper expression during the day.
We also examined dClk RNA, which cycles essentially antiphase to that of dper and is expressed at low constant levels in per-null mutants (6). Consistent with the impaired function of dPER(Δ), dClk transcript levels in wper01; per(Δ)-HAHis flies are relatively flat and pegged close to the trough values observed in the wild-type per+ transgenic flies (Fig. 7B). The essentially mirror image response of dClk expression compared to the dper RNA profile further indicates that PER(Δ) has a severely impaired ability to inhibit dCLK-CYC-dependent transcription.
Further evidence that dPER(Δ) is highly impaired in its ability to repress dCLK-driven transcriptional activation was obtained from ChIP assays. It was recently reported that the dCLK-CYC complex rhythmically binds to E-box containing circadian regulatory sequences that drive daily cycles in dper or tim expression, with the highest binding occurring at midday and lowest association during the late night/early morning (73). As expected, the temporal profile for dCLK interaction with the dper circadian relevant E-box element in wper01; per+-HAHis transgenic flies is very similar to that previously obtained with wild-type flies (Fig. 7C). However, in wper01; per(Δ)-HAHis transgenics, the relative binding of dCLK to the same circadian DNA element is high at all times in a daily cycle compared to what is seen for wild-type dPER-expressing transgenic flies. Together, our findings indicate that despite the stable association of dPER(Δ) with dCLK, the dCLK-CYC complex remains bound to circadian relevant promoters and is actively engaged in stimulation of transcription.
The first circadian clock protein to be characterized biochemically is dPER from Drosophila melanogaster (19). It was shown to undergo progressive changes in phosphorylation over a significant portion of the day, from hypo- to hyperphosphorylated species. Daily changes in abundance are temporally linked to the phosphorylated state of dPER, whereby the appearance of hyperphosphorylated species precedes rapid decreases in overall levels. More-recent work established that DBT is a major kinase controlling the phosphorylation and stability of dPER (35, 53) and that hyperphosphorylated variants of dPER are targeted to the 26S proteasome/ubiquitin pathway by the F-box protein SLIMB (26, 38). A highly shared mechanism operates in the mammalian system for the mPERs, whereby the mammalian homologs of DBT (CK1ε and the δ variant) and SLIMB (β-TrCP1 and possibly β-TrCP2) play major roles in the temporal regulation of mPERs' phosphorylation and stability (37). Herein, we identify the major or sole DBT interaction domain on dPER (termed dPDBD) that is required in vivo for dPER hyperphosphorylation, temporal instability, and repressor function. Similar results were also obtained by others using a slightly smaller internal deletion (51a). Moreover, our findings suggest that the dPDBD plays distinct roles in dPER degradation and repressor function and that dPER has a mode of action in transcriptional inhibition that is more indirect than previously thought (Fig. 8).
Earlier reports suggested that the amino-terminal half of dPER and possibly just the first 385 aa are sufficient to stably bind DBT (35, 36, 63). However, we show that in flies the absence of the dPDBD abolishes any detectable interaction between dPER and DBT (Fig. 6). Based on the effects of DBT binding to dPER(Δ) in S2 cells, which are less dramatic than those seen for flies (compare Fig. 3 and 6), it is possible that the use of high-level expression systems (35, 63) or a highly stable dPER-β-Gal fusion (36) in the earlier studies exaggerated weak interactions. Thus, while it is likely that there are other DBT binding regions on dPER (especially those that might be involved only in transient interactions), our findings indicate that in vivo the DBT-dependent hyperphosphorylation and degradation of dPER have an absolute requirement for the dPDBD, highlighting the tight association between hyperphosphorylation and enhanced degradation. These data are well accommodated by prior work showing that hyperphosphorylated isoforms of dPER are preferentially targeted by SLIMB (26, 38). Presumably, the ability of DBT to promote the hyperphosphorylation of dPER requires a stable and perhaps long-term association with the substrate, consistent with the observation that DBT is bound to dPER during a significant portion of the daily cycle (36). The tight interaction between dPER and DBT is also highlighted by the redistribution of predominately nucleus-localized DBT to the cytoplasm in pacemaker cells only during times in a daily cycle when dPER is present in the cytoplasm (36). Although the ability of CK1 to phosphorylate dPER in an in vitro reconstituted assay (Fig. 2C and data not shown) suggests that DBT and dPER directly interact, our data do not rule out the possibility that the stable interaction between DBT and dPER observed in vivo is stimulated or dependent on other factors that either directly or indirectly interact with the dPDBD.
Likewise, in the mammalian clockworks a region beginning around the middle of mPER1 and mPER2 and extending about 200 aa mediates their stable association with CK1ε (65, 67). Here too, removal of major CK1ε binding domains (CKBD) on mPER1 or -2 attenuates their CK1ε-driven instability and hyperphosphorylation (1, 22), although the physiological relevance of these regions has not been tested in animals. In contrast to mPER1 and -2, mPER3 displays no or weak interactions with CK1ε (1, 43). Rather, it is thought that the association of mPER3 with mPER1 enables mPER1 to act as a bridge and bring CK1ε into a favorable proximity to phosphorylate mPER3 (43). There is only limited knowledge of the structural features underlying CK1ε docking regions, with arguably the best characterized being the F-X-X-X-F motif present in the mammalian NFAT1 transcription factor (52). mPER1 and -2 but not -3 have this signature motif in their CKBD regions, and alteration of this motif greatly attenuates binding to CK1ε (52, 61). Intriguingly, when we did a BLAST analysis of our 57-aa region encompassing the dPDBD against the mPER1 and mPER2 protein sequences, it aligned within a subset of their CKBD that included the F-X-X-X-F motifs, but the alignment with mPER3 was noticeably dispersed (Fig. 9). While this finding is highly suggestive, the F-X-X-X-F motif is not found in the dPDBD, and future work will be required to more precisely define the key structural features.
Although the dPDBD promotes DBT-dependent hyperphosphorylation of dPER, it is unlikely to be a major site directly modified by phosphorylation. When the majority of Ser and Thr residues in this region (those conserved with dPER from other Drosophila species) are converted to Ala residues, the modified dPER undergoes progressive phosphorylation and degradation kinetics indistinguishable from those of the wild-type control version (Fig. 2D). This also seems to be the case for mammalian PERs. For example, a more detailed analysis showed that deletion of either of two small regions on mPER2 (aa 582 to 606 or aa 731 to 756) greatly reduced the ability of CK1ε to stably interact and promote degradation of mPER2 (22). However, recent work mapping phosphorylation sites on mPER2 did not identify either aa 582 to 606 or aa 731 to 756 as areas modified by phosphorylation (66).
Thus, although there might be structural features that differ between the DBT/CK1ε docking sites on mammalian and fly PERs, there are remarkable similarities. Most notable is the presence of one or a few small regions that are not major targets for phosphorylation but serve to stably bind DBT/CK1ε. Once stably bound, DBT/CK1ε either directly or indirectly (e.g., via other kinases) stimulates progressive phosphorylation at other sites until a threshold of multiphosphorylation is attained that enhances degradation by SLIMB/β-TrCP-mediated pathways (Fig. 8). Obviously, time-of-day-dependent variations in phosphorylation that regulate the levels of key transcriptional repressors constitute one mechanism that can control the timing and relative potency of repressor function, giving rise to cyclical gene expression.
In addition to regulating stability, DBT has been implicated in modulating the subcellular localization of dPER (e.g., references 7, 14, and 54), a function also ascribed to CK1ε in the case of mPERs (e.g., reference 67). We did not observe differences in the subcellular distributions between dPER(Δ) and wild-type dPER expressed in S2 cells, and whole-mount staining of adult Drosophila heads showed strong staining of dPER(Δ) in nuclei of dPER-expressing cells (data not shown), consistent with the exclusively nuclear staining of dPER in key pacemaker cells of mutants with abolished or severely reduced dbt activity (14). These results are in agreement with our biochemical studies showing that similar amounts of dCLK, which is the limiting component (5) and exclusively localized in the nucleus of both S2 cells (34) and fly clock cells (32), copurifies with dPER(Δ) and wild-type dPER (Fig. 3 and 6). Nonetheless, recent work by Nawathean and colleagues using an internal deletion smaller than ours suggests that the DBT binding domain enhances the nuclear localization of dPER (51a). The reason for this apparent discrepancy is not clear, but we note that by far the majority of recombinant dPER is found in the cytoplasm when expressed in S2 cells (e.g., references 10, 34, and 51). It is possible that in our S2 cell system the overall proportion of cytoplasmic dPER is higher, making it difficult to detect subtle differences in nuclear staining. Nonetheless, similar to the results obtained by Nawathean et al., when we placed a nuclear localization signal (NLS) on dPER and dPER(Δ), both proteins were highly localized in the nucleus with increased repressor function; however, dPER(Δ) was still approximately twofold less efficient in blocking dCLK-dependent transcription (data not shown). Although we cannot exclude possible effects on subcellular distribution, our results indicate that the strongly attenuated repressor function of dPER(Δ) is not explained simply by a lack of interaction with dCLK.
Why is the binding of dPER(Δ) to dCLK not sufficient for robust inhibition of transactivation activity? A possible answer is based on prior work suggesting that hyperphosphorylated dPER might be a more potent repressor of dCLK-CYC transcriptional activity (51), rendering the mainly hypophosphorylated dPER(Δ) deficient in this capacity. Another non-mutually exclusive possibility is based on our recent findings indicating that dPER is required for the DBT-dependent hyperphosphorylation of dCLK during the night/early day, events that promote the rapid degradation of dCLK and possibly reduce its transactivation activity (34, 73). Presumably, dPER can act as a bridge to enhance the phosphorylation of dCLK by DBT (73) (Fig. 8). Indeed, in per0 flies DBT is located largely in the nucleus of pacemaker neurons (36), yet hyperphosphorylation of dCLK is not observed (34, 73) (Fig. 5C). Our findings greatly strengthen this proposal by showing that dPER(Δ), which can bind dCLK but is impaired in its association with DBT, does not support the hyperphosphorylation of dCLK (Fig. 5C). A scenario where PER acts as a molecular bridge by which DBT can phosphorylate other clock proteins is similar to that proposed for mPER1 in stimulating CK1ε phosphorylation of mPER3 (43) and mCRYs (21), suggesting that PERs might have a general role in targeting DBT/CK1ε to numerous clock components. Even more impressive, a similar mechanism also occurs in the Neurospora clock, whereby the binding of CK1a (a CK1 homolog) to FREQUENCY is critical for inhibiting the transcriptional activity of the WHITE COLLAR COMPLEX (31). Thus, there is remarkable conservation of posttranslational regulatory pathways operating in widely divergent clocks.
Despite the reasonable possibilities that the dPDBD modulates dPER repressor function by regulating dPER phosphorylation and/or serving as a conduit to facilitate DBT-dependent phosphorylation of dCLK, there is circumstantial evidence from S2 cells and flies suggesting that neither scenario is obligatory for dPER to exhibit repressor capabilities. For example, in our S2 cell culture system, dPER(Δ) displays little repressor function compared to wild-type dPER even in the absence of exogenous DBT (Fig. 4), conditions under which only the hypophosphorylated isoforms of dPER(Δ) and wild-type dPER are observed (Fig. 2A) and in which more dCLK stably interacts with dPER(Δ) (Fig. 3C). Furthermore, in mutant flies with highly impaired DBT activities/levels, evidence suggests that dPER can still inhibit dCLK-CYC-driven transcription (14, 69), though the relative strength of this repression is not known. These results raise the possibility that the dPDBD acts as a docking site for other factors besides DBT that play a more direct role in inhibiting dCLK-CYC-dependent transactivation, e.g., activities involved in chromatin remodeling (8, 23) (Fig. 8). Indeed, the DBT-mediated phosphorylation of dCLK might be involved only in regulating dCLK stability whereas repression of its activity is carried out by other factors. However, it is important to note that in the aforementioned examples based on results obtained with cultured S2 cells or dbt-impaired mutants, the elevated levels of dPER might override stimulatory effects of phosphorylation and/or DBT on the ability of dPER to function as a transcriptional inhibitor. This reasoning might also explain why recombinant dPER was able to block dCLK-CYC binding to E-box containing DNA elements in an in vitro assay (41). Taken together, we speculate that dPER "alone" has low intrinsic repressor capabilities that are enhanced by the dPDBD-dependent assembly of DBT and/or other factors that more directly modulate the ability of dCLK-CYC to stimulate transcription.
Although future work will be required to determine if phosphorylation regulates the transactivation potential of dCLK, our findings highlight a clear qualitative difference in the roles of the dPDBD with regards to dPER instability and repressor function. Namely, whereas differences in the SLIMB-mediated degradation of wild-type dPER and dPER(Δ) are observed only under conditions of DBT-dependent hyperphosphorylation (Fig. 2), this is not the case for transcriptional inhibition (Fig. 4).
A more secondary mode of action for dPER as a transcriptional repressor is reminiscent of how mPERs are thought to function in the negative limb of the mammalian clockworks, where the mCRYs are the major inhibitors. Although mPERs exhibit some repressor capacity in cultured cell assays and they are found in complexes with mammalian CLK-BMAL1, it is likely that their main function in transcriptional regulation is to control the timing of mCRYs nuclear localization (42). This theme of roles as indirect repressors of circadian gene expression is also observed for FREQUENCY in Neurospora (59) and KaiC in cyanobacteria (64). A strikingly common feature of these indirect repressors is that they undergo complex daily changes in phosphorylation that are central to clock progression. Indeed, a daily rhythm in the cyanobacterial clock protein KaiC can be recapitulated in vitro using ATP and recombinant versions of the three critical clock proteins (KaiA, KaiB, and KaiC) from this system (50). While it is likely that the "minimal" circadian phosphorylation programs in eukaryotes are more complex, the bacterial work elegantly suggests that the most basic and ancient building block of circadian clocks is a biochemical oscillator based on time-of-day-specific phosphorylation of one or more key clock proteins. This central biochemical oscillator can be viewed as a semiautonomous subsystem from which temporal coordinates are largely transduced by phase-specific interactions with a variety of regulatory factors that subsequently control the activities of major transcription factors, leading to rhythmic gene expression and ultimately to daily changes in physiology and behavior.
We thank D. Chang and S. Reppert for providing a series of dper-containing plasmids that we used in our initial screen for regions that modulate dPER phosphorylation and stability in S2 cells. We thank Pipat Nawathean and Michael Rosbash for sharing unpublished results.
This work was supported by NIH grants awarded to I.E. (NS34958) and P.E.H. (NS052854).
- Received 15 December 2006.
- Returned for modification 2 February 2007.
- Accepted 11 April 2007.
↵▿ Published ahead of print on 23 April 2007.
- American Society for Microbiology


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Alfredo Serri: L'Universo si Ricompone nel Silenzio
October 2, 2014 – January 18, 2015
Alfredo Serri (Florence 1898-1972 ) was a musician and painter active in the Tuscan art scene of the 20th century, although he preferred not to publicize his work nor follow the lead of popular art "politics". This is the first retrospective exhibition of his work, entitled l'Universo si Ricompone nel Silenzio (the universe comes together in silence), now at the Ente Cassa di Risparmio di Firenze, curated by Stefano De Rosa.
On exhibit are some 100 paintings by the artist, many trompe l'oeil and still lifes, for the most part from a single private collection, in collaboration with the Galleria Open Art of Prato. Here, the viewer can appreciate these rarely seen works of art by the first student of artist Pietro Annigoni (1910-1988), painted by Serri after he decided to leave the world of music and dedicate his creative endeavors to painting. His works are now exhibited in numerous Italian and foreign art collections, public and private, including the Galleria d'Arte Moderna and the Roberto Longhi Foundation in Florence, the Civic Museum of Milan, the Fondazione Guelpa of Ivrea, and the Rockefeller Collection in New York.
Spazio Mostre Ente Cassa di Risparmio di Firenze
Via Bufalini 6, Florence – ITALY
Open: Monday – Friday 9am-7pm
Saturday and Sunday 10am-1pm and 3-7pm
Free entrance


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|Property TypeVacant Land
|Lot Size1.8 acres
Monopoly Realty
|Property TypeVacant Land
|Lot Size1.8 acres
R15//West St Paul/Last remaining lot in the development! Fabulous opportunity to build your dream home in a new subdivision in West St Paul located only a few minutes from the perimeter, but away from the noise. The developer's guidelines will ensure that your home is in good company. This 1.8 acre pie shaped lot is situated on a cul-de-sac backing onto Grassmere Creek. Roads have been paved and lots are ready to be built on. Hydro, gas, MTS & Shaw at the lot. (id:21591)
Listing Office: Monopoly Realty
Data Provided by Winnipeg REALTORS® Association
Last Modified :20/01/2020 11:23:03 AM


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RAC/RAS YHA series
Wall and floor mounted split systems
–Home is not
just a place,
it's a feeling.
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of fresh air.
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comfort and calm
–Choose from our range of air conditioners, designed to make you feel at home.
–If you have questions about this range, or any of our products, please get in touch.


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Sylish cameras are nothing new, but Nikon takes sexiness in cameras to a new level with their S50c. It's curvy, sleek, compact and feature-packed. It includes a huge 3.0″ LCD screen and offers 7.2 megapixel images. Is the smaller frame worth the slight lack of image quality?
Nikon is well known for producing high-quality digital cameras, most of them being point-and-shoots. Their collection is large, currently with 13 point-and-shoot cameras to choose from. Certainly, they like to give consumers choice. One area where Nikon lacks however, is that they haven't had a near-DSLR quality point-and-shoot for years, except the P5000 which was announced earlier this year. Canon holds the title there, as they have numerous high-quality P-n-S cameras to choose from.
What Nikon does excel at though, is creating the sexiest digital cameras on the market. Yes, I said sexy, and so did Nikon. There is a reason they have Kate Moss modelling off the S-series in their commercials. There's no denying the fact that it's a classy camera, and compact as well. It will fit into a purse, pocket, cleavage.. anywhere.
We first took a look at the S6 last October which is where this cameras style evolved from. Despite its small frame, it has curves in the right places, easy to hold onto and lots of things to love. Yes I am still talking about a camera.
The S50c as I mentioned, is part of Nikon's style series. First and foremost, cameras in this series have to look good. Second, they are not known for offering professionals a slew of options to deal with, but is made easy to use by anyone who wants to take some quick photos and look good while doing so.
Found here is a 2.5″ sensor capable of capturing 7.2 megapixels, aka 3072×2304. This is fantastic for printing, although it's probably overkill for most people to begin with. The lens is a 3x Zoom-Nikkor with a focal range of 6.3-18.9mm with an aperture of f/3.3-4.2. If digital mode is used, zoom is increased 4x, but the results are less than ideal.
One huge gripe I have with Nikon's point-and-shoots is that there is no viewfinder, instead they have a very large 3.0″ LCD screen. This is fine when conditions are right, but I took this camera to a park a few weeks ago and couldn't even see what was going on in the screen because of the sun. I am sure I am not alone here, it's impossible. If you are out in the sun, you can't see what's on the screen, it's common sense. That said, I'd love to see a viewfinder on a smaller Nikon point-in-shoot in the future. The P5000 has a viewfinder, but it's closer to a DSLR with a fixed lens than a point-and-shoot.
That rant aside, the screen has a nice 230,000-dot resolution, similar to what's found on their D-series. The S50c has a larger screen than what's available on those though. The camera has 13MB worth of internal memory and accepts SD cards for expansion. It accepts SDHC cards as well, in case you want to grab a card with a very high density.
ISO is the new megapixel. As the megapixel race is slowing down, the ISO rates are going up. The S50c offers a range of ISO 100 all the way up to 1600. Given the fact that this is a small camera in a modest price-range, I would not expect to see amazing results with anything above ISO 400.
Also included is vibration reduction, which when activated, will drain the battery far quicker. However, VR is an amazing technology that is an absolute joy to use if you are travelling without a tripod. The VR is built straight into the lens, not the body, a plus.
The rated battery life is 130 shots on a single battery charge. After the initial charge, I had the camera die on me after about 100 shots, so I didn't quite make their rated spec. From any viewpoint, 130 pictures per battery charge is horrible, but not uncommon of such small cameras. This is a side-effect of having a custom battery in such a small frame. If the camera utilized AA batteries, the shots/charge ratio would be at least five-fold.
Now that we have covered all the bases, lets now take a look at image quality, color and also our final thoughts.
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Nov 14, 2016 12:10:03 PM Edited Nov 14, 2016 12:31:07 PM by Valeria K
Apr 25, 2019 02:21:58 AM Edited Apr 25, 2019 02:37:06 AM by Goran V
Have the same problem "Financial transactions for Alexandr I**Edited for Community Guidelines** (15154190) have been limited. Please check your email for additional information or contact customer support."
can you help me with that?
Apr 25, 2019 02:38:46 AM by Goran V
Hi Alexander,
Our team has already reached out to you via ticket with more details about this. If you have any additional questions feel free to follow up on your ticket directly and our team will assist you further. You can access your tickets on this Link, thank you.
May 24, 2019 10:46:18 PM by Myra E
May 24, 2019 11:33:11 PM by Riri A
Hi Myra,
We checked your account for you and can see that you have an open ticket being handled by the team - https://support.upwork.com/requests/. We'll go ahead and follow up your ticket with the team, and feel free to reply on the same ticket thread so that they can assist you further.
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Global Online Sales EMEA Lead
Sam is the founding member of Snapchat's SMB team. Previously working with SMBs at both Google and Facebook, he brings a wealth of expertise on the digital ad landscape and the strategies that drive success across verticals. As Snapchat's EMEA SMB lead, Sam has vast experience helping clients of all shapes and sizes navigate new platforms and exceed their performance goals.


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Central Themes and Key Points
KEY POINTS IN DEVELOPMENTS IN EAST ASIA >> 20th CENTURY
Japan in China, 1937-1945
In the context of a) the political chaos that follows the fall of the centralized dynastic power of the Qing in the Republican Revolution in 1911 and b) the growing nationalism that crystallizes as the May 4th Movement after the 1919 Versailles Peace settlement — two political parties work and compete to reunify China and to modernize it to face the challenge of imperialist encroachment by the West and Japan. These are the Nationalist Party (Guomindang or Kuomintang) and the Chinese Communist Party.
Inadequate political control over the Japanese military, economic strains, and the worldwide Depression of the 1930s set the stage for the rise of the military in Japan and the pursuit of Japanese imperialist interests in Asia. Japan feels excluded by the West in the division of spoils in China. Japan pursues its own dominance of China by occupying Manchuria in 1931 and invading China in 1937 and remaining there until its defeat at the conclusion of WW II in 1945.
In China, the army of the Nationalist Party, led by Chiang Kai-shek (political heir of Sun Yat-sen), marches north in 1926 on the "Northern Expedition" from its base in southern China to establish a new government at Nanking in 1927 and to reunify part of China. This is sometimes called the Nationalist Revolution. The Nationalist government remained in power in Nanking until 1937 (1927-37 is known as the "Nanking Decade") when it is forced by the Japanese invasion to move inland and ultimately establish its wartime capital in Chungking (Chongqing) in 1938, where it remains until 1945. Japan captures the capital city of Nanking in 1937 in a brutal battle and subsequent reign of terror known as the "Rape of Nanking."
When the Japanese attack the American fleet in Pearl Harbor, Hawaii, on December 7, 1941, the United States enters World War II and goes to war with Japan; the war ends when the U.S. drops atomic bombs on Hiroshima (August 6) and Nagasaki (August 9) in Japan in 1945 and Japan surrenders unconditionally to the Allied forces. Japan's first attempt to enter the modern international system ends in failure.
During the course of the war Japan conquers other Asian nations, pursuing its own imperialist objectives and challenging Western powers for economic and military dominance in Asia. Hostility and unsettled issues resulting from the Japanese occupation remain in Japan's relations with Korea, China, and the countries of SE Asia.
When WW II ends in 1945 with Japan's defeat in China, the Nationalists and the Communist forces fight a civil war for control of China. The Communists are victorious in 1949 and the Nationalists leave the mainland of China and establish a rival government on the island of Taiwan. (The rival governments continue to exist today as the People's Republic of China on the mainland and the Republic of China on Taiwan.)
© 2019 Asia for Educators, Columbia University |


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Life is very unpredictable. We never know what will happen in the next moment. For example, say you're in the office, and a phone call comes and tells you that your spouse was severely injured in a traffic accident, or one of your parents had a heart attack. The next step is to make them go to the hospital and start treatment immediately. They also think of better treatment options as they do not like the limited hospital visits and the presence of many other patients.
At this point, you may come up with the idea of home doctor services. Home Doctors across Australia is offering medical services in the comfort of your own home. Priority is given to the right treatment, but economic efficiency is also essential to middle-class citizens. You do not want to spoil it in between when you take the patient home, but after a few days, you realize that the cost is off-budget, and return it to hospital.
Let's do a comparative analysis of the cost-effectiveness of home doctor services in advance to make the right decision. There are a few essential factors to keep in mind.
The first is the severity of the disease. If the severity of the illness is so high that it can be handled at home and all the necessary facilities (oxygenation, therapies, counseling, etc.) can be arranged at home, the patient can be taken for home treatment. If this is possible, the cost of treatment may be lower compared to hospitalization because of the hospital bed rent.
In the hospital doctor, the patient is examined at least twice a day, regardless of whether there is a need and the fee is added to the bill. However, if the patient is at home, the doctor can only come when needed or just for a regular check-up to see the improvement, and you only have to pay for these limited visits. However, when the severity is more significant, the home care option is not convenient.
There may be a need for advanced care. For example, assuming that the patient received initial treatment at the hospital, the doctor suggested extended treatment up to 3 months. In this case, it's very costly to bear the hospital costs for three months. The best option would be home nursing. Take all the doctor's instructions and, if necessary, appoint a nurse, look after them at home and see how they recover before you.
Think about the availability of home doctor services. If your home is in the heart of the city, all home-care services are always available when you call them. However, this is not the case if you are in a remote location and do not have easy access to all the necessary facilities. Now, if you want to choose Home Healthcare, you may need to pay more, double or triple the normal fees to get everything you need at home.


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Whoohoo! We are very happy to be the nonprofit of the month at Two22 Brew for the month of October. They donate $2.22 of every $10 in profit to local nonprofits. How cool is that?
We want to give a very special thank you to Kamryn Cobb, who kicked off our month at Two22 by selling goodies from Drizzle Bakeshop to raise money for Inside the Orchestra.
Go anytime this month to support our programs. We would also love to have you join us for Inside the Orchestra at Two22 on October 19th, when we will be there for the evening. We will have music, giveaways, information about our season, and more. Come out and hang with us.


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A clean and protected engine bay just finishes off a vehicle when having one of our detailing packages (the engine clean would be done before the detailing process).
Firstly we remove any excess debris and then cover any electrical components, air intakes and alarms to stop water damage. A strong degreaser/All Purpose Cleaner (APC) will be applied to the engine and the underside of the bonnet, which is then carefully agitated with various detailing brushes. The engine is then rinsed off, and a protective coating is applied to help it stay clean for longer.
On certain supercars and sports cars, it is possible vastly improve the appearance of an engine bay by (for example) machine polishing carbon covers and treating them with GTechniq coatings, re-painting tatty grilles to reverse the signs of aging and so on. If your car falls into this category, be sure to mention when booking if this would be of interest.
£65 - £400+
Time: 2 - 20 hoursAdd this to one of our packages
Unit 12
Railway Street Industrial Park
Gillingham Kent ME7 1YQ
+44 (0)1634 581560