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Home » Travel Blog » Top 6 best amusement park & best theme park in Asia
Top 6 best amusement park & best theme park in Asia
Summer vacation is a fun time for youngsters. Summer holidays are popular among families, with many opting for places that include theme parks or other forms of entertainment. In Asia, there are several possibilities to visit an international theme park and participate in interesting activities for both children and adults. Now, scroll down to see the top 6 finest amusement parks in Asia and Southeast Asia, as well as the greatest theme parks in Asia, for your ideal family holiday.
Singapore Universal Studios
Legoland, Malaysia. One of the best amusement parks in Asia.
Ocean Park, Hong Kong. Image by: best theme park in asia blog.
Siam Park City, Thailand
Siam Park City, Thailand. One of the best theme park in Asia.
Siam Park City has recently become very popular among Southeast Asean families. The park offers many interesting family activities such as swimming pools, roller coasters, and Thai massage, all at reasonable prices.
The Sea of Bangkok, the world's biggest artificial lake at 1,200 square meters, is located in Siam Park City. The Guinness Book of World Records recognized this lake. It gives the impression that you are swimming in the center of Bangkok. There are shallow places for youngsters with little waves, as well as a tumultuous Super Spiral portion. If the rough-and-tumble swimming makes your muscles hurt, you may pay 300 Baht for an hour of massage.
Siam Park City, Thailand. Image by: best theme park in asia blog.
Siam Park Water Park
Siam Park City is divided into five "worlds" for children: Water Park, Family World, X- Zone, Fantasy World, and Small World. Every year, 2 million visitors from all over the world visit the park.
With its thrilling activities for adults and children above 1.3m, X- Zone will take your breath away. Vortex, a huge roller coaster that reaches speeds of up to 80 kilometers per hour, is the most popular attraction. The three-spiraled Boomerang, the Mega Dance, and the 75-meter-high Giant are among the other rides. As you approach the park, don't be shocked if you hear screams and yells!
Siam Park City, Thailand. One of the best amusement parks in Asia.
If you are traveling with children, be sure to stop by Small World. This park is perfect for young families, featuring a Mini Carousel, Motorcycles, Mini Boats and Mini Astrofighters. The Small World castle is an ideal place to find shade and refreshment on scorching hot days. Family World—another section of the complex—features themed areas and attractions such as Dinotopia and Jurassic Adventure, African Adventure, and the Big Double Shock haunted house.
Siam Park City, Thailand.
The park is surrounded by restaurants and carts selling food, drinks and ice cream at reasonable prices. The entrance ticket to Siam Park City includes a lunch buffet featuring Thai, Indian and European dishes.
+ Address: 203 Suan Sayam Road, Khannayao District, Bangkok.
+ Entrance ticket (fee, prices): Adults: 30USD/day; children (under 1.3m): 25 USD/day.
More details can be found at www.siamparkcity.com/
+ How to get to Siam Park City: The fastest way to get to Siam Park City from central Bangkok is by taxi. The trip will take about 45 minutes and cost around 2000 baht. Remind the taxi driver to turn on the meter.
Legoland, Malaysia
Legoland, Malaysia. Image by: best theme park in asia blog.
Legoland Malaysia, located in Nusajnaya, Johor Bahru, is the first Lego Park in Asia. It opened in September 2012 and now offers 40 games, roller coasters, shows and activities for the whole family. The park is decorated with huge Lego structures designed and built by artists. The artists have constructed several of Asia's grandest sites, including the Great Wall, Angkor Wat and the Taj Mahal, using tens of millions of Lego blocks.
Previously, you had to fly to the USA, UK or Germany to explore this special type of park, but now there is one as close as Malaysia.
At Legoland you will have the chance to assemble your own Lego car and enter it in races. You can also see models of dinosaurs, or enter ancient Egypt at Pharaoh's World at the Land of Adventure. Star Wars fans will be delighted by the park's 2,000 Star Wars themed models.
Legoland, Malaysia.
If you want to cool off during a hot summer day, drop by Legoland Water Park, next to the theme park. The waterpark has more than 20 tube slides and 70 lego models. You can even "Build-A-Raft" from soft Lego blocks and float along the lazy river. You can also join the Aqua Zine Wave Racer water shooting game. Before you leave, stop by the Observation Tower to get a view of the whole Lego Complex.
Legoland, Malaysia. One of the best amusement parks in Asia
Legoland water park, Malaysia.
Visiting Legoland is very convenient, as you do not need to worry about finding accommodations. The Legoland Hotel is located right inside the park and features rooms decorated with Kingdom, Pirate and Adventure themes, a perfect place for children after an activity-packed day.
Theme room, Legoland, Malaysia.
+ Address: Legoland, 7, Jalan Legoland, Medini, 79250 Nusajaya, Johohr.
+ Entrance tickets (prices): Adult (12+): RM 196. Children: RM 156 for tickets booked online seven days in advance at www.legoland.com.my/book-visit/day-tickets.
+ How to go to legoland malaysia: From Kuala Lumpur, Terminal Bersepadu Selatan Station in Berjaya Times Square, you can take a bus to Johor Bahru (a 4–5 hour trip). More information about buses can be found at: www.legoland.com.my/planning-your-visit/how-to-get-here/bus-service.
Ocean Park, Hong Kong
Extreme thrill fans cannot miss Ocean Park in Hong Kong, which is famous for the Abyss Tower's 56 meter free fall, and the Hair Raiser, Hong Kong's fastest coaster reaching 88 km/h. The park also features 20 other coasters. Visitors to Ocean Park enjoy seeing cable cars cross the park between the Summit and Waterfront areas.
Ocean Park, Hong Kong.
The Park is 870,000 square meters and contains an indoor water park, an aquarium, a zoo and an amusement park. You and your family will have the chance to see marine animals such as jellyfish, dolphins, and sea dogs, and to learn more about marine ecosystems and environmental protection. You can also visit world-famous animals, like the panda couple An An and Jia Jia, the monkey couple Le Le and Hu Hu, and other rare species such as red pandas, giant iguanas and Chinese crocodiles.
Ocean Park offers three educational shows per day, featuring performances from dolphins and see dogs. The Birds Theatre allows audience members to interact with hunting birds like eagles and vultures, and the Arctic Wolf Den offers a comprehensive view of this species' lifestyle. The show ticket is included in the entrance ticket.
+ Address: Ocean Park, Aberdeen, Hong Kong. Tel: (852) 3923 2323
+ Entrance tickets (fees, prices): Adult (12+): $438. Children (3-11): $219.
+ How to get to Ocean Park: Ocean Park is open all days except for major holidays. To get the park, take bus #629 near MTR Admiralty (B Terminal),or bus #171 from Lai Chi Kok South Horizons to Aberdeen Tunnel Toll Plaza. More information can be found at: www.oceanpark.com.hk
+ Events: Ocean Park secured a prestigious Applause Award which "honors an amusement park whose management, operations and creative accomplishments have inspired the industry with their foresight, originality and sound business development."
Disneyland Tokyo, Japan
Tokyo Disneyland. Image by: best theme park in asia blog.
Mickey Mouse, Donald Duck, Snow White, and Peter Pan have been a significant part of many childhoods across many generations. A child's trip to Disneyland is a journey to paradise, and an adult's trip to Disneyland is a journey back to childhood.
Tokyo Disneyland.
Disneyland Tokyo is a part of the Disney brand, which includes Disneyland in California and Magic Kingdom in Florida. It is located in Urayasu, Chiba, East Tokyo (USA). World Bazaar, Tomorrowland, Toontown, Fantasyland, Critter Country, Westernland, and Adventureland are the seven themed zones of the park. A parade of popular cartoon characters takes place every morning, while a two-hour light display takes place every evening.
The DisneySea park, which is inspired by ocean myths, distinguishes itself from its competitors and draws the world's fourth highest number of park visitors.
The Mediterranean Harbour, which replicates Italy's Venice Harbour with beautiful waterways, the majestic MiraCosta Hotel, and delicate gondola boats, is located at the park's entrance. The Mysterious Island, inspired by Jules Verne's writings, stands in the heart of the park. You may join a voyage to the earth's core utilizing the most modern scientific vehicles here. Mermaid Lagoon, the Arabian Coast, and Port Discovery are all must-see sites.
Tokyo Disney Sea.
Between May and June, when the weather is pleasant and there are few people, is the perfect time to visit Tokyo Disneyland and DisneySea. Weekends are usually quite busy, and you may have to wait for many hours. The cost of a day at the park, including food, drink, and souvenirs, will be at least 10,000 yen.
+ Address: 〒279-0031 Chiba Prefecture, Urayasu, Maihama, 1−1
+ Entrance tickets: Adult's (18+) day ticket: 7,400 yen (18+); Juniors (12–17): 6,400; and children (4–11): 4,800. Details can be found at www.tokyodisneyresort.jp/en/ticket.
+ How to get to Disneyland Tokyo: The easiest way to reach Tokyo Disneyland is by train, which will take about 20 minutes from Tokyo central gas station to Maihama, right at the park entrance. More information can be found at: www.tokyodisneyresort.jp
Everland, South Korea
Everland, South Korea. Image by: best theme park in asia blog.
Everland entertainment park is Korea's first family park, located about one hour from Seoul. Global Fair, Magic Land, Zootopia, European Adventure, and American Adventure are the park's five themed lands. It may take your family many days to tour the entire region, as it is Korea's largest entertainment park.
Everland, South Korea.
Children who want to experience thrilling high speeds should go on the T–Express ride, which is 1779 meters long and the sixth longest wooden roller coaster in the world. It can reach a top speed of 104 km/h at a 77 degree angle . Those who love nature can explore the French-style four season garden that features more than 140 types of tulips, lilies (from June to August) and daisies (from September to October). Zootopia is home to wild animals from all over the world, such as horses, tigers, lions, elephants, penguins, and domestic animals like goats, sheep and dogs.
Everland is open all week with daily shows. On the weekends there are parades and concerts from famous music artists.
+ Address: 199, Everland-ro, Pogsok-eup, Cheoin-gu, Yongin-si, Gyeonggi-do.
+ Entrance tickets: 54.000 won/day for adult and 43.000 won/day for children.
+ How to get to everland: From Seoul, Gangnam station (Seoul metro number 2) at Gate 10, take the 5002 bus to Everland. Traveling time is 50 minutes . More details can be found at: www.everland.com/web/multi/english/everland/main.html.
Universal Studios, Singapore
Universal Studios.
The Sentosa Express train will transport you from the heart of Singapore to WaterFront station, right adjacent to Universal Studios, for just 35 Singapore dollars. The first movie theme park in Southeast Asia features seven zones and 24 exhibits based on Hollywood movies. There are eighteen of them that are exclusive to Singapore Park. Without having to go to the United States, you and your family will be able to see amazing sequences from the Mummy, Transformers, Jurassic Park, Shrek, and Madagascar.
Spaghetti Space Chase at Universal Studios Singapore. Image by: best theme park in asia blog.
Image by: best theme park in asia blog.
Hollywood Avenue, near the central lake, is the busiest part of Universal Studios. Costumed figures pose for photos with tourists at one end of the avenue. Don't miss out on the Louis NY Pizza Parlor's wonderful New York-style pizza and other delectable meals from KT's Grill. Visit The Lost World, which displays preserved Jurassic dinosaurs and insects, as well as water activities, if you're interested in the prehistoric.
Queues tend to be long at this park. If you don't have much time, prioritize visiting Transformers, Revenge of the Mummy, the Jurassic Park water world, and Far Far Away.
Don't forget about the nighttime shows. Wings of Time by the sea, Crane Dance, the world's largest puppet play, and the fireworks spectacular near Hollywood Avenue are among them. These performances, which usually run between five and twenty minutes, draw a large number of tourists. For the best seats, inquire about performance schedules and arrive early.
Credit: best amusement park in asia blog.
Wings of Time
+ Address: 8 Sentosa Gateway, Singapore 098269
More details can be found at: www.rwsentosa.com/Homepage/Attractions/UniversalStudiosSingapore.
+ Entrance tickets: Adults (13–59): S$74. Children (4–12): S$54
+ How to get to Universal Studio Singapore: From the center of Singapore, take the Sentosa Express to WaterFront Station, right at the entrance of Universal Studios.
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JVI Classic Spotlights
About JVI
Genome Replication and Regulation of Viral Gene Expression
Simian Virus 40 DNA Replication Is Dependent on an Interaction between Topoisomerase I and the C-Terminal End of T Antigen
Sujata Khopde, Daniel T. Simmons
Sujata Khopde
Daniel T. Simmons
DOI: 10.1128/JVI.01314-07
Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.
The replication of simian virus 40 (SV40) DNA has been extensively investigated as a model to understand the replication of eukaryotic DNA in detail (4, 13, 23, 46). SV40 DNA has a well-defined origin, and it encodes an initiator protein called large T antigen that functions as a bidirectional helicase and, in coordination with host proteins, promotes bidirectional replication from the viral origin (28, 54, 65, 66). The use of cell-free SV40 DNA replication systems has identified more than 10 host proteins that are essential for this process (31, 53, 62, 67, 68). These proteins have been purified and investigated for their role in initiation of DNA synthesis and elongation of DNA (21, 26, 33, 36, 43, 56, 69). Among them, only three proteins, DNA polymerase α-primase (pol/prim), replication protein A (RPA), and topoisomerase I (topo I), are required for initiation of DNA chains by forming an active initiation complex with T antigen (8, 12, 18, 20, 35, 55).
In order to initiate replication, T antigen binds to the core origin in a defined manner (9, 10, 25, 32, 39, 64). In the presence of ATP, T antigen assembles at the core origin from individual monomers into a double hexamer or as a preformed hexamer followed by cooperative association with another hexamer (9, 58). The double hexamer is the functionally active form of T antigen that untwists the AT-rich tract, melts the early palindrome, and begins to unwind DNA bidirectionally using its helicase activity (1-3, 11). The three cellular initiator proteins are then recruited to the origin, but the dynamics of this process are not clearly understood. In a recent study (47), we showed evidence that pol/prim must be present in order for topo I and RPA to bind, suggesting that it binds first. That study also suggested that topo I is recruited next, followed by RPA. The function of topo I is to continually relieve the superhelical tension generated during the DNA-unwinding process (69). We have assumed that this activity is tightly coordinated with DNA unwinding when topo I and T antigen interact with one another (48-50). RPA is required to stabilize the unwound single strand and to stimulate pol/prim activity (16, 24). The primase subunit of pol/prim triggers the formation of small RNA primers on the exposed single-stranded template, and these are extended into short stretches of DNA by polymerase α (38, 57). All three cellular proteins can directly bind with T antigen, and these interactions appear to be essential to effectively initiate DNA synthesis (12, 19, 35, 37, 48). However, exactly how all four proteins associate with one another to form an initiation machine is still not clearly understood.
Topo I can bind to T antigen in vitro, and multiple studies have provided evidence that this association is functionally important (18, 42, 48-50, 55). Topo I significantly enhances T-antigen-mediated DNA replication and induces the formation of much larger amounts of completed molecules (50, 55). The enzyme is needed from the point of initiation (18, 55) and cannot stimulate replication if added after the reaction has started (55). It makes the T antigen unwinding reaction origin specific when tested with a linear DNA fragment (50). Under DNA replication assay conditions, topo I binds preferentially to a double-hexameric structure of T antigen associated with origin DNA and cannot readily interact with smaller structures (14). The stoichiometry of this complex is one molecule of topo I per T-antigen hexamer (14, 47).
Our previous studies (44, 48) have demonstrated the existence of two binding sites for topo I on T antigen, one at the N-terminal region (residues 83 to 160) and the other at the C-terminal region (residues 602 to 708). We also showed that the cap domain of topo I interacts with both sites (44). Electron microscopic analysis of T-antigen double hexamers assembled over the core origin revealed that the two hexamers communicate through the N-terminal region that includes the origin binding domain (residues 131 to 260) (59-61). X-ray crystallography of origin binding domain dimers identified the putative sites of interaction between adjacent hexamers (34), in agreement with earlier genetic data (64). One topo I binding site must therefore be located close to the hexamer-hexamer interface, and the other must be near the distal ends of the double hexamer (44). Since the recruitment of topo I to T-antigen double hexamers is also dependent on an association with DNA (47), this suggests that topo I is attracted to the C-terminal site (where the DNA is exposed) to participate in initiation of DNA replication. Separation of the duplex DNA is thought to take place very close to the central channel at the back end of the double hexamer (15, 51), and by associating close to that site, topo I might work together with T antigen as a swivelase, relaxing the DNA as it becomes unwound (14, 44). The role of the N-terminal topo I binding site on T antigen is not known, but since it is located close to the binding site for RPA (63), it is possible that the topo I enzyme there might be involved more directly in DNA synthesis as opposed to DNA unwinding.
In the present study, we sought to more precisely identify the topo I binding site at the C-terminal end of T antigen and investigate its role in SV40 DNA replication. For this purpose, we generated a number of T-antigen mutants with substitutions within this region and tested them for their ability to bind topo I and to support DNA replication. We identified a cluster of amino acids on the surface of the T-antigen helicase domain that is needed for binding to topo I and for promoting DNA replication. The results strongly indicate that an interaction of that region of T antigen with topo I is required for the proper functioning of the DNA replication machine.
Site-directed mutagenesis.Single point mutations were introduced into SV40 T antigen cDNA using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. Mutations were generated in a recombinant plasmid consisting of the baculovirus transfer vector p1393 (BD Pharmingen) with an SV40 T-antigen cDNA insert (6). Briefly, two synthetic oligonucleotide primers containing the desired mutation were annealed to the denatured plasmid DNA template, and the primers were extended by using Pfu Turbo polymerase in a PCR. Nonmutated parent DNA was cleaved by digestion with DpnI for 1 h at 37°C, and the DNA was introduced into XL-Gold competent cells (Stratagene). The DNA was purified from individual clones and sequenced using appropriate primers to confirm the mutation. The same mutations were generated in p1393 with a cDNA insert that expresses only amino acids 246 to 708 of T antigen (48). Some mutations were introduced into pBS/SV40 (30) by the same method. This plasmid contains the entire SV40 genome inserted into the BamHI site of pSK(+) (Stratagene).
Generation of recombinant baculoviruses.Sf9 (Spodoptera frugipenda) cells were cotransfected with p1393-T-antigen constructs and BaculoSapphire DNA (Orbigen) to produce recombinant baculoviruses as described by the manufacturer. T-antigen-expressing recombinant viruses were detected by immunofluorescence with anti-T-antigen monoclonal antibody PAb101 (17). The virus was amplified twice by infecting Sf9 cells (2 × 107 cells each time) before proceeding with protein purification.
Protein expression and purification.Wild-type (WT) and mutant full-length T antigens were purified by immunoaffinity chromatography from infected High 5 insect cells using PAb101 antibody-coupled Sepharose 4B beads as described by Mastrangelo et al. (32). T-antigen fragments (Tfs) consisting of residues 246 to 708 were purified by first binding to PAb101 beads, followed by high-pH elution as described by Simanis and Lane (45). All full-length as well as fragment T antigens were dialyzed overnight against a solution containing 0.01 M Tris (pH 8.0), 0.1 M NaCl, 0.001 M EDTA, 0.001 M dithiothreitol, and 50% glycerol and stored at −20°C. Protein concentrations were estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using bovine serum albumin (BSA) as a standard.
Topo I was purified from infected High 5 insect cells as previously described (52).
Origin DNA unwinding assays.32P-labeled origin-containing 64-bp DNA was generated from the following two oligonucleotides as described by Jiao and Simmons (22): top strand, 5′CACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTA 3′; bottom strand, 5′TAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTG 3′. For the unwinding assay, 400 ng of full-length T antigen was incubated with 2 ng of radiolabeled 64-bp DNA for 1 h at 37°C in unwinding buffer (30 mM HEPES [pH 7.5], 7 mM MgCl2, 40 mM creatine phosphate, 4 mM ATP, 1 mM dithiothreitol, and 0.1 mg/ml BSA) supplemented with 50 μg of creatine phosphokinase per ml and 2.8 μg of Escherichia coli single-stranded DNA binding protein (Pharmacia) per ml. The reactions were terminated by addition of stop buffer (0.02 M EDTA, 0.4% SDS, and 0.25 mg proteinase K per ml [final concentrations]), and the mixtures were further incubated for 30 min at 37°C. The reaction mixtures were subjected to electrophoresis on nondenaturing 7% acrylamide gels for 3 h at 110 V. The gels were dried and exposed to phosphor screens overnight, and the labeled DNA was visualized and quantitated with a Molecular Dynamics PhosphorImager.
In vitro DNA replication assays.SV40 DNA replication assays were performed in topo I-deficient Ad293 cell extracts as previously described (54, 55). Topo I-deficient extracts were identified by their responsiveness to added purified topo I. The reaction mixtures contained 236 ng of origin-containing pSVO + 11, 200 to 300 μg of cell extracts, and 1 μg of full-length T antigen in the presence or absence of 100 ng purified human topo I. DNA synthesis was terminated by incubation with stop buffer (0.02 M EDTA, 0.4% SDS, 0.25 mg of proteinase K per ml [final concentrations]) for 30 min at 37°C. The labeled DNA was purified by phenol chloroform extraction followed by passage through a Spin 20 column (Princeton Separation). The DNA was separated on 1.5% agarose gels (in Tris-borate-EDTA) by electrophoresis for 5 h at 100 V. The gels were dried and exposed, and the labeled DNA products were quantified with a PhosphorImager.
ELISAs.Enzyme linked immunosorbent assays (ELISAs) were performed as described elsewhere (48). In brief, 100 ng of topo I was allowed to attach to wells of microtiter plates. After blocking with phosphate-buffered saline containing 1% gelatin, 0 ng to 200 ng of Tf (residues 246 to 708) was added and incubated for 2 h at room temperature. After the unbound protein was washed off, an antibody specific to the C-terminal end of T antigen (PAb101) was added to all wells, followed by the addition of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG). Peroxidase substrate (ortho-phenylenediamine) was then added and left for a few minutes, and the reaction was terminated by the addition of 50 μl of 2.5 M H2SO4. The absorbance of each well was measured at 490 nm using a Dynex plate reader. The absorbance was plotted against the corresponding concentration of the Tf, and the data were analyzed using the Graph Pad Prism 5 program (Graph Pad Prism Software Inc., San Diego, CA).
Far-Western blotting assays.Far-Western blotting assays were carried out as described by Simmons et al. (48). Six hundred nanograms of topo I was loaded in each well of an SDS-10% acrylamide gel and subjected to electrophoresis for 50 min at 200 V, followed by transfer to a nitrocellulose membrane for 1 h at 100 V. The membrane was cut into strips corresponding to each lane, and the membrane strips were blocked with milk buffer (3.5% dry milk in Tris-buffered saline containing 0.3% Tween 20). Each strip was incubated with 1.2 μg of Tf consisting of residues 246 to 708 in 2 ml of milk buffer for 2 h at room temperature. After the excess protein was washed off, the membrane strips were incubated with PAb101 antibody and ECL reagent (Amersham) according to the manufacturer's recommendations. As a negative control, BSA was used instead of T antigen. Bands were visualized by chemiluminescence and quantified using a FluorChem 8800 instrument (Alpha Innotech).
Virus replication assays.pBS/SV40 containing various mutations was cut into two pieces with BamHI, diluted, and end ligated to generate circular SV40 DNA. A 0.5-μg amount of ligated DNA was transfected into 3 × 106 BSC-1 monkey cells using DEAE dextran as previously described (30). Virus was collected 4 days later by repeated freeze-thaws, and the titer of the released virus was determined by plaque assays on BSC-1 cells. Cells were stained on day 12 with neutral red, and plaques were counted.
Generation of T-antigen mutants defective in binding topo I.The C-terminal topo I binding site in T antigen is located between residues 602 and 708 (44). This C-terminal region is believed to lie on the face of the distal surfaces of double hexamers that form over the SV40 origin (59, 60) and appears to attract one topo I molecule per hexamer (14). In this study, we mutagenized certain residues within this region to more narrowly map the site of interaction and to determine the significance of the binding of topo I to this region for SV40 DNA replication. We chose to mutagenize certain residues within a smaller region (residues 602 to 627) based on the fact that deletion of residues beyond residue 627 had no influence on DNA replication activity (29, 41). In the first round of mutagenesis, the residues that were mutated were determined by sequence conservation among various polyomavirus T antigens (Fig. 1A) as well as by their exposure on the surface of the helicase domain (27). Only one mutant in this series (K616R) was found to be defective in binding topo I (Table 1). A second series of mutants was then generated based on the modeling of T-antigen-topo I complexes using the docking program Z-dock (http://zlab.bu.edu/zlab/people.shtml). Multiple hits were obtained, but we selected the thermodynamically most stable model that predicted the involvement of residue K616 in T antigen and the cap region of topo I. This model is illustrated in Fig. 1B. All T-antigen residues in close proximity to and facing topo I in the model as well as other exposed residues were selected for mutagenesis (Fig. 1B and Table 1). The list includes two residues (K550 and Y552) (Fig. 1A) that map to the same face on the protein but lie outside of region from residue 602 to 627.
Sequence and structural analysis of the C-terminal topo I binding site of SV40 T antigen. (A) Sequences of various T antigens from five different polyomaviruses, i.e., monkey SV40, human BK virus (BKV) and JC virus (JCV), monkey lymphotropic papovavirus (LPV), and mouse polyomavirus (PyV) from residues 602 to 627 (SV40 numbering) and residues 550 and 552 (as compiled by James M. Pipas). Mutated residues are shown in bold, and the substitutions are indicated in bold italic at the bottom. (B) Close-up view of the T-antigen-topo I interaction site predicted by a Z-dock model of the trimeric T-antigen helicase domain-topo I complex. A T-antigen monomer (blue, red, and green) is associated with the cap domain of topo I (residues 215 to 433) (yellow). The residues of T antigen mutated in this study are shown in red and green. The green residue corresponds to K616.
Summary of biochemical properties of T-antigen mutants
The specific substitutions that were made at these various residues were determined in part by sequence conservation (Fig. 1A), in part by the expected impact of various substitutions on T-antigen structure, and in part by the prediction that a certain substitution would alter topo I binding. In most cases, conservative mutations were selected; however, in some cases, more dramatic changes were made in order to prevent possible interactions between the two proteins. We made several substitutions at residues 616 and 621 (Fig. 1A and Table 1). Mutations were generated in two baculovirus transfer vectors: one that contains the cDNA for full-length T antigen and another that expresses a truncated T antigen (residues 246 to 708) missing the N-terminal domain. Recombinant baculoviruses were made, and the mutant proteins were expressed and purified from virus-infected insect cells. Yields of all mutant full-length and fragment proteins were equivalent to those obtained with the WT proteins.
Origin-unwinding activity of mutant T antigens.Since all the mutations we made were at residues that were exposed on the surface of the helicase domain, we considered it unlikely that the mutations would significantly alter the overall structure of the protein or interfere with its major function as an origin-specific helicase. To check this, however, we tested the mutant proteins for their ability to unwind a synthetic 64-bp origin-containing DNA. The separated labeled "top" strand of this DNA was detected and quantitated by gel electrophoresis (Fig. 2A). All mutant proteins were able to unwind the origin DNA like WT T antigen (Fig. 2B and Table 1). Most mutants possessed 80 to 100% of the normal activity, although some of them (e.g., K605Q, N618S, and G622A) appeared to be slightly more active than WT T antigen. Therefore, all mutant proteins possessed at least normal origin-unwinding activity. This conclusion was reinforced by assays with "minicircular" origin-containing DNA performed as previously described (14). In this assay, the formation of underwound 388-bp circular DNA is dependent on both T antigen and topo I. All mutants were tested and found to possess normal activity (data not shown).
Origin-unwinding assay of WT T and mutant T antigens. Full-length WT or mutant T antigen was incubated with 32P-end-labeled 64-bp origin DNA in the presence of ATP for 1 h at 37°C. The DNA was purified and applied to a 7% acrylamide gel. (A) A representative gel result with WT T antigen and several mutants is shown. The positions of the labeled double-stranded (ds) and separated labeled single-stranded (ss) DNAs are shown. Lane 1 shows the results of a reaction without T antigen, and lane 2 shows the labeled single-stranded DNA obtained after boiling the labeled 64-bp DNA. (B) Bar graph of the origin-unwinding activities of all studied T antigen mutants as measured by the released labeled single-stranded DNA. Activity is expressed as a percentage of that of WT T antigen. Error bars represent standard deviations obtained from three data sets.
In vitro DNA replication activity of mutant T antigens.Topo I is required for efficient SV40 DNA replication (49, 55, 65, 68, 69). Previous reports (49, 50, 55) have shown that the addition of topo I to T-antigen-mediated replication stimulates replication and greatly promotes the completion of DNA chains. The enzyme acts at the point of initiation and is a component of the initiation complex (18, 47, 55). To check whether the T-antigen mutants can promote DNA replication and whether topo I stimulates replication, we performed SV40 DNA replication assays using topo I-deficient 293 cell extracts. In this assay, topo I (100 ng) stimulated total DNA replication in the presence of WT T antigen by about 2.5-fold, although the synthesis of covalently closed form I molecules was stimulated by 7- to 8-fold (Fig. 3 and Table 1), in agreement with earlier results (55). Mutant T antigens promoted DNA replication to various degrees (Fig. 3 and Table 1). Some mutants, (e.g., Q613R and N618S), behaved similarly to the WT (Fig. 3B and C). Others, (e.g., K616M and M621I), demonstrated a partial defect in DNA replication, but replication was only slightly stimulated by topo I. A third category of mutants (e.g., Y612N and K616R), supported virtually no DNA replication, and added topo I had no effect (Fig. 3B and C). Different substitutions at the same site had different effects. Replacement of Lys 616 with the similarly charged Arg or with polar Gln resulted in a complete replication defect, whereas replacement with the hydrophobic Met resulted in only partial loss of activity (Fig. 3 and Table 1). Likewise, replacement of Met 621 with Ile permitted a modest stimulation of replication by topo I (Fig. 3C and Table 1), whereas replacement with Ser led to the total absence of stimulation. When the fold stimulation was measured only in completed form I molecules, the differences between the activities of certain mutants and WT T antigen were even more striking (Fig. 3D). In this case, mutants clearly fit into only two categories: those that were stimulated normally by topo I and those that were completely refractive to added topo I (Fig. 3D and Table 1). These results strongly support the idea that the interaction of topo I with certain T-antigen residues within this region is required for full DNA replication activity.
DNA replication activity of WT and mutant T antigens in the presence or absence of topo I. (A) A representative gel of DNA replication assays performed in the presence or absence of added topo I is illustrated. As a control in lane 1, neither T antigen nor topo I was added to the 293 cell extract. Labeled synthesized DNA was separated on a 1.5% agarose gel and detected with a PhosphorImager. The positions of replicative intermediates (RIs), covalently closed circular relaxed DNA (CCC), and supercoiled form I DNA are shown. (B) Bar graph of replication assays performed with all mutant T antigens in the presence or absence of added topo I. Replication activity is expressed as a percentage of that of WT T antigen in the absence of topo I. (C and D) Bar graphs of the fold stimulation obtained by the addition of topo I as measured by total labeled DNA (C) or labeled form I DNA only (D). A line across y = 1 in both graphs represents no stimulation. Error bars represent standard deviations from three data sets.
Binding of T-antigen mutants to topo I. (i) ELISAs.Since some of the mutant T antigens failed to support DNA replication in the presence of topo I, we were interested in testing them for their ability to bind topo I. Because of the presence of another topo I-interacting site near the N-terminal region (residues 83 to 160) (44), we introduced the same mutations in a Tf consisting of residues 246 to 708 and missing the N-terminal binding site. These mutant fragments were first tested for their topo I binding activity by ELISAs. Different amounts of Tfs were added to topo I-coated wells, and the bound T antigen was detected with an antibody (PAb101) to the C-terminal end of T antigen (17), horseradish peroxidase-conjugated anti-mouse antibody, and substrate. As previously described (48), binding between the two proteins occurs readily and reaches saturation at about 100 ng of T antigen (Fig. 4A). Several of the mutant fragments bound to topo I less well than the WT Tf, as shown in the representative ELISA in Fig. 4A. To quantitate the signal and compare binding of topo I to various T-antigen mutants, we used the Prism 5 data fitting program (Graph Pad Software Inc., San Diego, CA) to first calculate a 50% effective concentration (EC50) for each curve. This value corresponds to the concentration of the Tf at which the absorbance is half of maximum. We then calculated the reciprocal of this number and normalized each to the value for the WT Tf, which was taken as 100%. These numbers are plotted in Fig. 4B and included in Table 1.
ELISA between topo I and Tfs (residues 246 to 708). One hundred nanograms of topo I was incubated with various amounts (0, 6.25, 12.5, 25, 50, 100, and 200 ng) of WT or mutant Tfs (designated f) in ELISA plates. The plates were washed and incubated with T-antigen-specific antibody PAb101, followed by the addition of horseradish peroxidase-labeled anti-mouse IgG and peroxidase substrate. The reaction was terminated by addition of 2.5 M sulfuric acid and detected by absorbance at 490 nm using a plate reader. Optical density (O.D.) at 490 nm was plotted against the corresponding concentration of Tfs. The data were fitted to a nonlinear one-site binding equation using the Graph Pad Prism 5 program (Graph Pad Prism Software Inc., San Diego, CA) (solid lines in panel A). (A) Representative graph with some of the mutants. (B) An EC50 value (defined in the text) was calculated from each curve. The reciprocal of the EC50 was calculated for WT and mutant Tfs and expressed as a percentage of that of WT Tf. Error bars represent standard deviations obtained from three data sets.
Mutant fragments Y612Nf, F617Nf, and K616Rf bound poorly to topo I compared to WT Tf (Fig. 4B and Table 1), whereas mutants K616Qf, K616Mf, M621If, M621Sf, K550Qf, and Y552Nf bound a little better to topo I but still significantly less well than WT Tf. Interestingly, the topo I binding-defective mutants were also DNA replication defective, whereas those that bound topo I normally supported replication normally (Table 1). Overall, there was an excellent correlation between the two activities.
(ii) Far-Western blotting assay.To corroborate the ELISA findings, we performed far-Western assays where purified topo I was first subjected to SDS-polyacrylamide gel electrophoresis, transferred to a membrane, and then incubated with T-antigen mutant fragments. Binding was detected by reactivity with monoclonal antibody PAb101 (17), horseradish peroxidase-conjugated anti-mouse antibody, and chemiluminescent substrate. As shown in the representative gel in Fig. 5A, fragments bound to the topo I on the membrane to different extents. The signal was quantitated and plotted as a percentage of that of the WT T fragment (Fig. 5B). In agreement with the ELISA results, the L603Vf, K605Qf, Q613Rf, N618Sf, G622Af, and I623Vf mutants were as competent as WT Tf in binding topo I, whereas mutants K616Rf, F617Nf, and M621Sf were about 30% efficient in binding topo I. These data agreed with the ELISA results for all mutants except Y612Nf, which showed close to 60% binding by far-Western blotting but only 12% by ELISA (Table 1). This is a large difference, but it appears that this mutant T antigen, as well as all other binding-defective mutants listed above, bound better to denatured topo I in the far-Western assay than to native topo I in ELISAs. These differences indicate that the binding site on the denatured topo I is more tolerant of changes in T antigen than is the one on native topo I.
Far-Western blotting assay between topo I and Tfs (residues 246 to 708). Six hundred nanograms of topo I was applied to an SDS gel and transferred to a nitrocellulose membrane. Membrane strips were incubated with 1.2 μg of WT Tf or various mutant Tfs. Binding was detected with antibody PAb101, horseradish peroxidase-conjugated anti-mouse IgG, and chemiluminescence reagent. (A) Representative blot with some of the mutant proteins. As a control, BSA was incubated with a topo I membrane strip and is shown in lane 1. (B) The signals for WT and mutant Tfs were quantitated using a FluorChem 8800 instrument and plotted as a percentage of that of WT Tf. Error bars represent standard deviations obtained from three data sets.
Replication of mutant viruses in vivo.To investigate the significance of the T-antigen-topo I binding reaction for virus replication in vivo, we introduced several of the same mutations in pBS/SV40 (30), a plasmid that contains the entire SV40 genome. BSC-1 monkey cells were transfected with mutant or WT plasmid, and after 4 days, the virus was collected and the titer was determined by plaque assays. DNA replication- and topo I binding-defective mutants Y612N, F617N, and M621S yielded significantly less virus (>2,000-fold-, 14-fold-, and 5-fold-lower titers, respectively), and the plaques that appeared for M621S were much smaller (about one-third) than normal. On the other hand, DNA replication- and topo I binding-normal mutant L605Q gave rise to normal amounts of virus, and plaque size was not affected. These results indicate that T-antigen mutations that reduce the binding to topo I interfere with normal virus replication in vivo.
Localization of the C-terminal topo I binding site on T antigen.Our previous mapping studies proposed the existence of two binding sites for topo I on T antigen, one involving amino acids 83 to 160 (N-terminal site) and the other amino acids 602 to 708 (C-terminal site) (44). By a mutagenesis strategy, we identified the specific amino acids within the C-terminal site that are responsible for binding topo I. Mutants that we made with substitutions at residues 612, 616, 617, and 621 were mostly defective in topo I binding, indicating that these residues are important for contact with topo I. Two other mutants with changes at residues 550 and 552 were also defective at binding topo I (Table 1). Although these two residues lie outside of the linear region (residues 602 to 708) previously identified by fragment analysis (44), they map close to the other four residues on the surface of the protein (27). These six residues identify a patch on the edge of the back surface of the helicase domain that is responsible for binding topo I (Fig. 6). This region is highlighted in each of the subunits of the hexamer structure (Fig. 6A). The patch consists of charged, polar, and hydrophobic amino acids; however, based on the substitutions that are more tolerated at residues 616 and 621 (Table 1), it appears that hydrophobic interactions may play a significant role in the binding between these two proteins.
Locations of the T-antigen residues involved in binding topo I and a model of the topo I-T-antigen complex. (A) Back view of the hexameric structure of the helicase domain of SV40 large T antigen (27), looking into the central channel. Residues K550, Y552 Y612, K616, F617, and M621 are highlighted in dark blue on each monomer. (B) A portion of the structure marked by the rectangle in panel A is expanded, and the highlighted residues are labeled. (C) Side view of a T-antigen hexamer with three front-facing monomers displayed in different colors (red, green, and purple). Residues K550, Y552 Y612, K616, F617, and M621 are shown in blue on these three monomers. In a double hexamer, the highlighted residues would be on the back edges. (D) Trimeric T antigen in the topo I-T-antigen model obtained from the Z docking program (http://zlab.bu.edu/zlab/people.shtml) was modified to contain a complete T-antigen hexamer associated with the cap domain of a single topo I molecule using the Swiss PDB viewer program. Each chain is displayed in a different color. The monomer of T antigen associated with topo I is marked in yellow, and topo I is in cyan. The structure shows the T-antigen central channel (at a slight angle) and the topo I cavity through which DNA passes.
The interaction between topo I and the C-terminal topo I binding site on T antigen is essential for SV40 DNA replication.All full-length mutants possessed normal origin-unwinding activity (Fig. 2) and could form normal amounts of double hexamers at the origin (data not shown), indicating that the mutations did not interfere with T antigen's major function as a helicase. In spite of this, several mutants were defective in promoting SV40 DNA replication in cell extracts and in responding to added topo I in stimulation of DNA synthesis (Fig. 3). The replication-defective mutants correlated precisely with those that were defective in topo I binding (Fig. 4 and 5 and Table 1). This strongly indicates that the binding of topo I to the C-terminal binding site in T antigen is required for optimal DNA replication.
Mutant T antigens supported SV40 DNA replication to various degrees; however, it was noticed that mutants that were severely compromised in DNA replication activity (e.g., K616R, F617N, and M621S) were also the ones that responded very poorly, if at all, to added topo I (Fig. 3 and Table 1). Mutants that displayed a partial loss of activity, (K616M and M621I) were stimulated slightly by added topo I, and those that had near-normal DNA replication activity were stimulated by topo I just like the WT (Fig. 3B). There was also a correlation between total levels of DNA replication and topo I binding activities of individual mutants. Hence, the mutants severely compromised in topo I binding were the same ones that were most defective in DNA replication, and those that had intermediate activity in one assay displayed an intermediate activity in the other (Table 1). These data all point to the conclusion that topo I binding is intimately linked with the ability of various mutant proteins to support DNA replication. Our in vivo replication data suggest a role of the binding of topo I to this end of T antigen in virus multiplication in culture as well.
According to our working model (47), during DNA replication in the presence of pol/prim, topo I is recruited by T antigen after it has formed a complete double hexamer over the origin (14), and this is followed by the binding of RPA to make an initiation complex (47). We now have evidence, presented in this paper, that topo I binds at least to the C-terminal site to participate in DNA replication. The N-terminal topo I binding site appears to be fully functional in the full-length mutant proteins as determined by topo I binding assays with these proteins (data not shown). However, its role in DNA replication is not known.
The major function of topo I during DNA replication is to relieve the supercoiling strain on unreplicated portions of the DNA (69). We have therefore proposed that topo I associates with the C-terminal binding sites within the context of a double hexamer to coordinate the processes of DNA relaxation and DNA unwinding (48-50). It has been shown (7) that a relative of SV40 T antigen (E1) encoded by papillomavirus 16 stimulates topo I activity. The E1 protein also has two binding sites for topo I, and it is the C-terminal site that most effectively stimulates topo I relaxation activity. It is reasonable to suggest, therefore, that a helicase-topo I association is needed for DNA replication for both virus families. However, in light of the fact that none of our T-antigen mutants were impaired in their ability to generate underwound circular DNA in the presence of topo I (data not shown), our current hypothesis is that this interaction is needed primarily for initiation of new DNA chains and not for relaxing unreplicated supercoiled DNA.
Stoichiometric analysis (14) showed that there are two molecules of topo I per double hexamer in the T-antigen-DNA-topo I complex; hence, one molecule of topo I is situated on each hexamer. In Fig. 6A, we highlight the binding sites on each monomer in the hexamer structure (27), and in Fig. 6B, we show an expanded view of one subunit. Each binding site for topo I consists of a number of residues that cluster to the external edge of each monomer of the hexamer. These sites are widely separated from one another on adjacent subunits (Fig. 6A and C). Hence, in double hexamers, the binding sites map to the distal edges of this complex (Fig. 6A and C). Modeling of the T-antigen hexamer and topo I complex by Z-dock (Fig. 6D) shows that the topo I molecule is located on the side of each hexamer, not in line with the double hexamer as might be assumed from the location of the binding sites. Consequently, the bound topo I molecule is at a large distance from the central channel of the helicase (Fig. 6D). Surprisingly, this channel and the groove through which DNA passes in topo I are nearly parallel to one another. Given the stoichiometry of binding (14, 47), it is most likely that topo I can bind to only one T-antigen subunit at a time. Considering the spatial relationship between the two proteins in this model and the fact that the DNA must be threaded through topo I as a double strand and through the T antigen helicase central channel at least as a single strand, we can infer that the DNA must be significantly bent as it connects the two proteins. This idea is consistent with studies that showed that topo I preferentially interacts with and nicks bent DNA (5, 40). There is no way of predicting whether topo I binds to only one subunit at all times or whether it interacts with different subunits at different times during the process of fork movement. It is certainly conceivable that the topo I enzyme switches from one monomer to another during this process, and if so, the attached DNA would therefore gyrate around as topo I moves from monomer to monomer.
Assembly of a topo I-T-antigen complex on DNA.There is good evidence that major structural rearrangements occur in T antigen as it assembles into a double hexamer over the origin (59, 61). Furthermore, complexes consisting of 11 subunits or fewer and origin DNA bind topo I much less well than fully formed double hexamers (14). The initial binding of topo I to T antigen must depend, therefore, on extensive rearrangements of subunits as they assemble into a double hexamer. We have previously proposed that topo I first engages the DNA and then attaches to the ends of the double hexamer (47). The results presented here suggest that as topo I associates with a binding site on one of the T-antigen monomers, the DNA becomes severely bent.
In summary, the evidence presented in this paper clearly points to a dynamic interaction between topo I and the back edges of the T-antigen double-hexamer helicase machine to initiate DNA replication and to promote the synthesis of complete daughter DNA molecules.
This work was supported by PHS grant CA36118 to D. T. Simmons.
We are indebted to Rupa Roy for technical assistance.
Received 15 June 2007.
Accepted 2 November 2007.
↵*Corresponding author. Mailing address: Department of Biological Sciences, University of Delaware, Newark, DE 19716. Phone: (302) 831-8547. Fax: (302) 831-2281. E-mail: dsimmons{at}udel.edu
↵▿ Published ahead of print on 14 November 2007.
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Tsurimoto, T., and B. Stillman. 1991. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J. Biol. Chem. 266:1961-1968.
Uhlmann-Schiffler, H., S. Seinsoth, and H. Stahl. 2002. Preformed hexamers of SV40 T antigen are active in RNA and origin-DNA unwinding. Nucleic Acids Res. 30:3192-3201.
Valle, M., X. S. Chen, L. E. Donate, E. Fanning, and J. M. Carazo. 2006. Structural basis for the cooperative assembly of large T antigen on the origin of replication. J. Mol. Biol. 357:1295-1305.
Valle, M., C. Gruss, L. Halmer, J. M. Carazo, and L. E. Donate. 2000. Large T-antigen double hexamers imaged at the simian virus 40 origin of replication. Mol. Cell. Biol. 20:34-41.
VanLoock, M. S., A. Alexandrov, X. Yu, N. R. Cozzarelli, and E. H. Egelman. 2002. SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes. Curr. Biol. 12:472-476.
Waga, S., and B. Stillman. 1994. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature (London) 369:207-212.
Weisshart, K., P. Taneja, and E. Fanning. 1998. The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication. J. Virol. 72:9771-9781.
Weisshart, K., P. Taneja, A. Jenne, U. Herbig, D. T. Simmons, and E. Fanning. 1999. Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding. J. Virol. 73:2201-2211.
Wobbe, C. R., F. B. Dean, Y. Murakami, J. A. Borowiec, P. Bullock, and J. Hurwitz. 1987. In vitro replication of DNA containing either the SV40 or the polyoma origin. Philos. Trans. R. Soc. London B 317:439-453.
Wobbe, C. R., F. B. Dean, Y. Murakami, L. Weissbach, and J. Hurwitz. 1986. Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains. Proc. Natl. Acad. Sci. USA 83:4612-4616.
Wobbe, C. R., L. Weissbach, J. A. Borowiec, F. B. Dean, Y. Murakami, P. Bullock, and J. Hurwitz. 1987. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc. Natl. Acad. Sci. USA 84:1834-1838.
Wold, M. S., D. H. Weinberg, D. M. Virshup, J. J. Li, and T. J. Kelly. 1989. Identification of cellular proteins required for simian virus 40 DNA replication. J. Biol. Chem. 264:2801-2809.
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Journal of Virology Jan 2008, 82 (3) 1136-1145; DOI: 10.1128/JVI.01314-07
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Inside Tina Turner's secret surgery
6 Oct, 2018 2:17pm 2 minutes to read
Blair Tuke and Usain Bolt buddy up
7 Oct, 2018 7:00am Quick Read
Tim Minchin donation writes new story for Auckland youth charity
7 Oct, 2018 5:00am
Ngā Rangatahi Toa performers rehearse their Manawa Ora production. Photo / Nick Reed
By: Dionne Christian
Arts & Books Editor, NZ Herald
dionne.christian@nzme.co.nz
A donation from Australian superstar Tim Minchin will allow a charity that works with youth at risk to expand its creative-arts-based programmes into South Auckland schools.
Started nine years ago, Ngā Rangatahi Toa (NRT) uses social justice and arts-based-programmes to connect with young people excluded from school and encourage them to stay in school or vocational training.
Minchin, the award-winning musician, comedian, actor and composer who wrote Matilda the Musical, chose NRT as the charity which will receive money from sales of tickets to Auckland concerts in April.
NRT executive director Huia O'Sullivan says Minchin's promoters provided an estimate of the amount which she won't reveal other than to say it's "not to be sneezed at".
"It will go back into the projects we run and, next year, we plan to roll out a partnership project with schools in South Auckland," says O'Sullivan. "It's an amazing gesture from Tim Minchin and we're just really blessed that he, as an artist, can understand and see the importance of having access to the arts for young people who may not otherwise be able to get involved."
Australian composer, musician and comedian Tim Minchin is donating money to local charities. Photo / Nick Reed
Minchin told the West Australian newspaper that giving money away is part of being lucky.
"It's saying to the wealthier of my ticket-buyers ... you get the best seats in the house and all the money goes to charity, so you get a good feeling as well. It is important that people like me do our little bit to promote the idea that lucky people should have social responsibility."
In Wellington, Arohanui Strings-Sistema, which provides free music classes vulnerable children across the cities northern suburbs, will receive money as will Australian charities in the cities Minchin performs in through March.
O'Sullivan believes he picked NRT after speaking with Auckland Live, custodians of the Herald Theatre where NRT performs its annual Manawa Ora showcase. This sees youth paired with top New Zealand musicians, actors, writers and artists to create public performances of music, movement and monologue.
Because the Aotea Centre is closed for renovations, Manawa Ora 2018 is part of the Basement Theatre's spring programme. Now in its sixth consecutive year, mentors Laughton Kora, Cat Ruka, Suli Moa and Tama Jaram have worked with 15 teenagers on "Invisible Threads", the theme for this year's showcase.
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It explores how young people navigate relationships and create connection by reflecting on the invisible threads that tie them to self, whānau and community.
"These connections, through reflections that form the invisible threads, are the transferable skills our rangatahi [young people] are able to replicate into their own lives, strengthening their protective factors and enhancing their resilience as they find their place in their whānau, community and world," O'Sullivan says. "Our role is to create the space for them to have their collective voices heard, shared and echoed."
What: Manawa Ora: Invisible Threads
Where & when: Basement Theatre Studio, Thursday, October 11 — Saturday, October 13
What: Tim Minchin Old Songs, New Songs, F ... You Songs
Where & when: Aotea Centre, ASB Theatre, Sunday, April 14 and Monday, April 15; Wellington's Michael Fowler Centre, Thursday, April 18 and Friday, April 19.
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Get the Cupcakes out and blow up the balloons, as the Stampers Showcase share everything Birthday themed. This month we are delivering a Birthday party we hope you'll join in with. Get your dancing shoes on and be ready to bop and hop.
If you've come to the party having passed the lovely Lori on the way, then welcome, you appear to be hopping already. I have no doubt that Lori has already shared some fabulous Birthday inspiration for you.
Me? …. I'm packing a punch with colour for this card and going all out Primary colours. I've been wanting to use the Layering Alphabet Dies for a while and finally had a play. Oh it was fun! The design of these dies makes lining the letters up so easy. There is a coordinating Stamp Set called Lined Alphabet, but I didn't feel this was needed here.
These dies are an investment piece that would earn you free Sale-a-Bration items right now. You could add the amazing Painted Seasons Bundle of Stamp Set and papers to your basket for FREE if you were to purchase these.
Happy I was indeed when I'd sorted out the rainbow colour order and lined the word happy up, but it needed more.
Like all disco party balls, we needed sparkle and stars, so turned to the Twinkle Builder Punch for these. With three different sizes of stars, it was a great way of using up scraps of glimmer paper. I literally scattered them right across the card and even added a couple to the bottom of the twine bow tails.
So we have colour, we have sparkles and we're happy, but what about the Birthday? A Retro feel was beginning to emerge, unless that was me reminiscing of my youth spent in Disco's? The Labeler Alphabet Stamp Set offers a real Retro vibe that helped complete the front of the card. My relatives used to add the year inside their cards when there wasn't an age on it. With this in mind, I added 2019 to the inside. Not everyone's cup of tea, but if kept, it's lovely to look back on in years to come.
Well, I hope you've enjoyed my Birthday share and that you'll join me in heading over to see what the lovely Kate has in store for us. Having partied with her in Orlando at the 30th Celebration of Stampin' Up! back in November, I feel sure you're in for a Birthday treat.
Want these Dies, Stamp Set and Punch for just £99? Want on going discount on your orders? Then speak to me about buying the bumper Starter Kit this month. Receive £170 worth of products for just £99 and receive a wealth of opportunities, friendships and a minimum of 20% on going discount.
Be sure to get in contact or convert your Wish List to a Starter Kit today by clicking on the Join my Team link below.
Oh Miss mikaela, this card is so fun and festive! And I just love your introduction!!! So exciting! hehe. Thanks tons for hopping with us, luv!
That is too cute! love the fun colors and those die cut letters are fantastic!
OMG… adore this!!! I've got to pull out those darn dies!!! You made it look AMAZING!


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Published: 2 May 2016
Imprint: Penguin Classics
Little Black Classics
Mighty Ape Paper Plus The Warehouse Whitcoulls Fishpond The Nile
Find local retailers
Arguing that a state's political power should lie with its people, these pieces from The Social Contract inspired the French revolution.
'No true Democracy has ever existed, nor ever will exist.'
In this selection from The Social Contract, Rousseau asserts that a state's only legitimate political authority comes from its people.
One of 46 new books in the bestselling Little Black Classics series, to celebrate the first ever Penguin Classic in 1946. Each book gives readers a taste of the Classics' huge range and diversity, with works from around the world and across the centuries - including fables, decadence, heartbreak, tall tales, satire, ghosts, battles and elephants.
Pub date: 2 May 2016
Peter Constantine's honors include the PEN Translation Prize, the National Translation Award, the Helen and Kurt Wolff Translation Prize, and Greece's Translators of Literature Prize. He translated Machiavelli's The Prince for Vintage Classics.
Jean-Jacques Rousseau was born in Geneva in 1712. He was a writer and political theorist of the Enlightenment. In 1750 he published his first important work 'A Discourse on the Sciences and the Arts' (1750) where he argued that man had become corrupted by society and civilisation. In 1755, he published 'Discourse on the Origin of Inequality' and in 'The Social Contract' (1762) he argued, "Man is born free, and everywhere he is in chains". This political treatise earned him exile from his home city of Geneva and arguably inspired the French Revolution (his ashes were transferred to the Pantheon in Paris in 1794). He also wrote 'Èmile', a treatise on education and 'The New Eloise' (1761). This novel scandalised the French authorities who ordered Rousseau's arrest. In his last 10 years, Rousseau wrote his 'Confessions'. In The Confessions he remembers his adventurous life, his achievements and the persecution he suffered from opponents. His revelations inspired the likes of Proust, Goethe and Tolstoy among others. Rousseau died on 2 July in France in 1778.
Also by Jean-Jacques Rousseau
David Wallace-Wells
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Jess Phillips
James Arvanitakis
Asia's Cauldron
Robert D. Kaplan
Moral Clarity
Susan Neiman
The Master Strategist
Ketan J Patel
The Road To 1945
A Citizen's Guide To Impeachment
Barbara Radnofsky
Bruce Bartlett
Mary Katharine Ham
The Israeli Solution
The Unholy Trinity
What We Do Now
Listen, Yankee!
Tom Hayden, Ricardo Alarcón


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Golfers of the Week
NCAC Championships
77 °f Thunderstorm In Vicinity
DU Hires Jason Badecki As Head Golf Coach
Denison head golf coach, Jason Badecki
Story Courtesy of Denison Sports Information Department
Denison University Associate Vice President and Director of Athletics Nan Carney-DeBord has announced the hiring of Jason Badecki as the new head men's and women's golf coach and assistant professor in the department of Health, Exercise and Sport Studies. Badecki becomes the first full-time head golf coach in the history of both programs.
"I am extremely honored and blessed to have the opportunity to lead both golf programs at Denison," said Badecki. "I have witnessed the reputation Denison has in the world of college athletics and when I visited campus it became clear those successes were not by chance. The vision that Nan has for the entire department and the passion she has to help her coaches give their student-athletes a first-class experience is incredible. I am excited to get to work and begin building both programs into the championship programs that I believe they can be."
"We are thrilled to welcome Jason Badecki to the Denison community," said Carney-DeBord. "Jason's previous experiences as a head coach of both genders at Eastern set him apart from a strong applicant pool. We are confident that he has the knowledge, passion and leadership abilities to lead the Big Red golf programs to even greater heights."
"I would also like to acknowledge the tremendous work by head men's golf coach Rodney Butt and head women's golf coach, Rick Bailey. Both have spent the last seven years as head coaches in part-time roles but never wavered in providing exceptional mentorship to their student-athletes."
Badecki comes to Denison after four years as the head women's golf coach at Eastern University (Pa.) and one season as the head coach of the men's and women's golf teams at Eastern. Badecki has guided the women to a pair of Middle Atlantic Conference championships in 2014 and 2017. Both conference titles resulted in appearances in the NCAA Division III Golf Championships. Badecki was named the MAC Women's Coach of the Year in 2014 after guiding the Eagles to their first NCAA postseason appearance. He picked up his second MAC Coach of the Year award earlier this year after capturing their second MAC championship. The 2017 Eagles women's golf team went on to finish 20th at the NCAA Division III Championship.
In four seasons leading the women's golf team, 13 players earned all-conference accolades and six were named to the Women's Golf Coaches Association All-American Scholar team.
The 2016-17 season marked Badecki's first as the head coach of the men's program. Eastern placed second in the MAC Freedom conference standings.
In addition to his role as head men's and women's golf coach at Eastern, Badecki also served as a staff advisor to their Student-Athlete Advisory Council and was the facilities manager for the athletic department.
Badecki is a 2012 graduate of Rutgers-Camden where he was a member of the golf team from 2010-12, He received a bachelor's degree in Marketing and in 2016 he completed his master's degree in business administration from Eastern University.


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sam angert
Midwood Veteran Returns Home After 2 Years In Limbo
rrojas
Sam Angert (right) and friend Lt. Joseph Forton, who was killed in the explosion. (Source:NYDailyNews.com)
A Midwood veteran who was severely injured while honorably serving his country in Iraq is finally going home after waiting 28 months to be medically discharged by the U.S. Army.
"It's been a waiting game for a long, long time," said Sam Angert in the New York Daily News. "I just wanted to start my life over."
Angert was told it would take six to eight months for his discharge, but in reality it took 28 months and that was only because Congressman Jerrold Nadler's office got involved.
"After a terrible and debilitating injury, I am so thrilled that Sam will now be able to end his two-year limbo and, at long last, return home to his family and future," said Nadler.
In August 2009, Angert was involved in an explosion that gravely injured him and claimed the life of his friend.
Angert's vehicle was struck by a improvised explosive device, leaving him with pieces of shrapnel in his face and arm. Also, there was severe hemorrhaging from when a piece of steel pierced his skull, sending him into a 19 day coma. After regaining consciousness, he went to Warrior Transition Unit at Fort Belvoir in Virginia to recover.
Overall, Angert has had four cranial surgeries, overcoming predictions that he would never be able to walk again. He plans on attending college and major in psychology, hoping to help out other wounded veterans like him.
"I've been fighting for freedom in our country," said Angert. "Now I just want to go live it."
Do you know a local veteran who recently returned from service abroad? Let us know!


Need Drug Rehab in Clifton, CO?
We understand getting help can be the most daunting aspect of rehabilitation.
More all-encompassing drug/alcohol rehabilitation centers encourage changes on a deeper level and aid their patients to completely alter their way of living, which is commonly very important to have long-lasting results. What is ordinarily seen in rehabilitation from addiction to drugs or alcohol that is continuing are: continued outpatient rehabilitation, recovery at the local facility, detoxing thoroughly with clinical oversight, and treatment at home. This works best when together with a balanced home situation. For those who are able to be part of a long-term rehabilitation center, we strongly recommend that you go for it.
Understanding the most powerful skills and life guidelines like helpful fear and stress handling skills, life coping techniques, and how to define reasonable personal boundaries in relationships, will significantly raise the likelihood of a sustained recovery with minimal regression.
We will help you learn these vital techniques so that we can give you the highest odds for a complete rehabilitation.
We can offer you a real path to recovery. We are more than happy to discuss your personal needs and plan for your path to recovery. Our specialists treat drug and alcohol addiction with the most powerful substance addictions treatment systems. We will be there arming you with the best structures and methods all throughout your rehab period, so you can handle the obstacles you will confront.
There are some substance abuse recovery programs are for even 4-5 months. Because everyone is different (and circumstances differ), there are drug and alcohol addictions recovery centers that are one month long processes, but most last actually longer. 90+ day drug treatments tend to have the longest lasting results. When a lot of people think of drug/alcohol abuse treatment, they commonly presume a month-long process. Nevertheless, it could depend on the drug recovery facility.
Substance abuse is a hurtful affliction that changes mind and body. Accordingly, our team of professionals have to address the 2 respective elements of recovery. Rehabilitation from addiction to drugs or alcohol begins with doing a detoxifying. We start focusing on the the mind when the drugs or alcohol are entirely eliminated.There is little chance for long term recovery until we manage and attend to any original causes for the addiction. Drug/alcohol addiction rehabilitation requires major changes in style of living and way of life. Often, addiction happens along with mental health issues, trauma, or behavioral disorders.
For the Clifton, Colorado area drug rehabs just give us call us today at our help-line (toll-free) Talk to one of our agents to look over your particular needs, finances, budget, and opportunities, along with ways we can help, as well as finding you a nearby a rehab facility that is best suited to you.


Skin cancer happens when skin cells change into abnormal cells and grow at an uncontrolled rate.
There are two main types of skin cancer: melanoma and non-malignant melanoma skin cancer and the number of cases for both is increasing worldwide.
Melanoma is a type of skin cancer in the cells that produce pigment – called melanocytes.
What are non-malignant melanoma skin cancers?
Non-malignant melanoma skin cancers develop among cells in the upper layers of the skin. The two most common types of non-malignant melanoma skin cancer are squamous cell carcinoma (SCC) and basal cell carcinoma (BCC).
Non-malignant melanoma skin cancer can occur anywhere on the skin, though it's most commonly found on parts of the body that have experienced long-term sun exposure – like the head, face, neck, arms, legs and back of the hands.
What are basal cell carcinomas?
Basal cell carcinomas usually stay in one place. However, they can sometimes spread to other nearby areas.
Basal cell carcinomas rarely spread to lymph nodes or other sites in the body.
What are squamous cell carcinomas?
Squamous cell carcinomas usually stay in one place. However, they can sometimes spread to other nearby areas.
Some high-risk squamous cell carcinomas have the potential to spread to lymph nodes or distant sites in the body.
Malignant melanoma is skin cancer. It develops from cells called melanocytes. Melanocytes make melanin, which gives skin its colour. Melanin also protects us from ultraviolet (UV) radiation which causes sunburn.
There are two main types of skin cancer – non-malignant melanoma and melanoma – along with some much rarer types. Non-malignant melanoma is more common, and it's much less likely to spread.


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Dulcene Hardin Miller
July 1, 1916 - August 28, 2020
Dulcene Hardin Miller was born in Flatrock, Illinois, to Wanda Holladay (Ferriman) and Ross C. Hardin on July l, 1916, and died August 28, 2020, at 104 years old. She was preceded in death by her parents, Wanda Holladay Ferriman and Ross C. Hardin, and her brother, Ross Hardin, Jr. She also was preceded in death by her husband, Richard H. Miller, in January 1998. Dulcene is survived by step-daughters: Sylvia Miller Lloyd, Radford, VA, and Cheryl Miller Rogers (Neil), Williamsburg, VA; step-son, Richard Kent Miller (Amanda), Pearisburg, VA; step-grandchildren: William Shelburne, III, Susan Shelburne Hunt (Reed), Angela Medley Hill (Scott) and Richard Earl Irwin-Miller; four great-grandchildren, Michael and Matthew Hill and Reagan and Jillian Hunt; and half-sister Ann Hardin Register (Jim), Daytona Beach, FL. Dulcene retired in 1978 after working 36 years as a Contract Administrator for the Defense Construction Supply Center (DCSC) for the U.S. Department of Defense. Before working for DCSC, she held several other jobs. Dulcene was a hard-working woman who held many responsible positions. She was a member of St. Joseph Cathedral parish and Columbus Mennerchor. In 1934, she graduated from Columbus East High School. Dulcene said she was blessed with many close and dear friends, especially Jeannine Palmer and the caring staff at The Forum at Knightsbridge and said, "Thanks for being there for me." Mass of Christian Burial will be at 10:30 AM on Wednesday, September 9, 2020 at St. Timothy Catholic Church, 1088 Thomas Lane, Columbus, Ohio 43220. Arrangements entrusted to Schoedinger Northwest Chapel.
Dulcene Hardin Miller was born in Flatrock, Illinois, to Wanda Holladay (Ferriman) and Ross C. Hardin on July l, 1916, and died August 28, 2020, at 104 years old. She was preceded in death by her parents, Wanda Holladay Ferriman and Ross C.... View Obituary & Service Information
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May 13 Briefing: How Well Are Seniors Making Choices Among Medicare's Private Plans And…
May 13 Briefing: How Well Are Seniors Making Choices Among Medicare's Private Plans And Does It Matter?
The typical Medicare beneficiary this year has 18 private Medicare Advantage plans and 35 stand-alone Part D drug plan options to consider, in addition to traditional Medicare. Medicare encourages seniors to make informed decisions with respect to their health coverage options when they first become eligible for Medicare, and to review these options annually so they select coverage that best meet their needs. Yet research suggests only a small share of Medicare beneficiaries voluntarily switch plans during Medicare's open enrollment periods. This raises questions about the role of choice in Medicare: How do Medicare beneficiaries choose among coverage options? Are Medicare beneficiaries happy with their coverage or just "sticky" when it comes to plan choice? What motivates some to switch plans and why don't others do so? What are the implications for Medicare beneficiaries, private plans, health care providers and Medicare's future?
At 9:30 a.m. ET Tuesday, May 13, the Kaiser Family Foundation will hold a policy briefing to examine how seniors and younger Medicare beneficiaries are negotiating their Medicare Advantage and Part D coverage options and consider the implications for them and for Medicare in the future. Swarthmore College Professor Barry Schwartz, author of The Paradox of Choice and a TED conference speaker, will provide opening remarks on why Americans value choice and the effects of abundant choice for individuals and their decision-making. His remarks will be followed by perspectives from an expert panel that will focus on Medicare, comprising Joe Antos, scholar at the American Enterprise Institute; Gretchen Jacobson, associate director for Medicare policy at the Foundation; Joshua Raskin, managing director at Barclays Capital; and Judith Stein, executive director of the Center for Medicare Advocacy. Foundation Senior Vice President Tricia Neuman will moderate.
WHEN: Tuesday, May 13, 9:30 a.m. to 11:00 a.m. (Registration and breakfast at 9 a.m.)
WHERE: Barbara Jordan Conference Center, Kaiser Family Foundation Offices 1330 G Street, NW, Washington, D.C. (one block west of Metro Center)
View the archived webcast of this briefing.
KFF: for trusted information on the health issues facing the nation and its people. The Kaiser Family Foundation is a nonprofit organization based in Menlo Park, California.
Tiffany Ford Fields
(202) 347-5270	|	tfordfields@kff.org
Medicare Beneficiaries' Out-of-Pocket Health Care Spending as a Share of Income Now and Projections for the Future
Serious Illness in Late Life: The Public's Views and Experiences
Medicare Advantage: How Robust Are Plans' Physician Networks?