text:
Welcome to ÜberProAudio - your definitive source for all things Pro Audio. Learn about cool music gear, instruments, studio recording tips, musicians, and much more! Checkout our famous "Who Plays What" section to find out the news on what your favorite musicians use for gear in their rigs.
Think you know something about guitar gear? Take our:
Guitar Gear Quiz Or Metal Gear Quiz Or Guitar Pedal Quiz - New!
Read more...
We try to provide a great starting point for your research, but we can't always get all the info. Post a comment and help us keep things up-to-date and relevant for the global music community!
Peter Cetera is a singer, songwriter, bass guitar player and producer best known for being an original member of the rock band Chicago, before launching a successful solo career.A lot of you have been begging us to research Cetera's Bass rig, so guess what...we did.Let's look at what gear and equipment has been seen in Peter Cetera, Chicago's Legendary Bassist's, Bass Rig.
Click the gear images for more info and specs at Musician's Friend
BassesNotes: Cetera tried many basses, the Fender P-bass being his favorite.
- '64 Fender Precision (featuring a rosewood fingerboard and custom Paisley-painted body. It now is painted White)
- Lake Placid Blue '65 Jazz Bass
- '64 Hofner Beatle Bass
- '65 Vox Constellation IV bass- Tune Bass Maniac- McPherson acoustic bass guitar
Basses of the past (Chicago):- Gibson EB-3- Rickenbacker 4001- Gibson Ripper- fretted and fretless Fender Jazz Basses- Spector Bass- Music Man StingRay
- Ibanez Roadstars- Steinberger
Bass Amps- Kustom- Acoustic- Sound City- Phase Linear- Orange- Ampeg rigs- Ampeg B-15
Strings and picks- Cetera began with La Bella flatwounds but moved on to roundwounds, never quite liking them as much as the flats. He now uses the Flatwounds.
- Fender medium picks


text:
20th Annual Messiah Sing-Along
The Frederick Children's Chorus will present the 20th annual Messiah Sing-along at the Weinberg Center for the Arts. Performers on stage will include a thirty-voice chorus, twenty-piece orchestra, and four soloists, led by Judith DuBose, Artistic Director. In the audience will be several hundred singers, waiting for their turn to sing, and listeners who have the opportunity to sit among the singers. Each year for eighteen years an enthusiastic audience has gathered, ready to sing the well-known choruses of Messiah by G. F. Handel.
In a sing-along, the orchestra on stage and the chorus on stage guide the audience to sing choruses like, "For Unto Us a Child is Born" and "His Yoke is Easy". The audience members report that they look forward to singing what is probably the most well-known of the choruses; "Hallelujah". Messiah is the most frequently performed major work world-wide. Messiah Sing-along concerts are prevalent throughout the United States, Europe and Asia.

subset: commoncrawl


text:
The Intermarkets Portfolio delivers over 1 billion page views and reaches more than 28 million unique visitors every month. Whether youre on one site, or on the ...
Spark : Real Traffic System. A Premium Advertising Network. SIGN UPLOG ... Copyright © 2010 Spark Studios. 10811 Washington Boulevard, 4th Floor Culver ...
Intermarkets is leading in Top 10K Sites, Top 100K Sites and Top 1M Sites.
Spark Studios has better usage coverage in more websites categories. Including Arts & Entertainment, Computer & Electronics, Software, Games and 16 other categories.
Intermarkets is leading in News & Media, People & Society, Religion & Spirituality, Law & Government and 5 other categories.
Spark Studios is leading in most countries, including France, Germany, Indonesia, Mexico and one other country.
Intermarkets is leading in United States, Italy, Pakistan and United Kingdom.


text:
Fruit Cake Bon Bons
Kimi Gaines
My husbands mom made these for years and sent them to us. I loved them so much but could never get the recipe from her. I found this recipe on cooks.com. After I found it her recipe arrived in the mail. Actually I like this recipe better. It is easier and less preparation. These do not last very long in our house.
- 1/2 c
- unsulphured molasses
- 1/4 c
- water
- 15 oz
- pkg of raisins
- 1 lb
- container of mixed candied fruit
- 1/2 c
- butter
- 2/3 c
- sugar
- 3
- eggs
- 1 c
- plus 2 tbsp sifted all-purpose flour
- 1/8 tsp
- baking soda
- 3/4 tsp
- cinnamon
- 3/4 tsp
- nutmeg
- 1/4 tsp
- allspice
- 1/4 tsp
- ground cloves
- 1/4q c
- orange juice or rum
- 1/2 c
- chopped nuts
1Blend together molasses and water in a large deep saucepan. Place over low heat; stir constantly until mixture comes to a boil. Add raisins; bring to a boil again. Reduce heat and simmer 5 minutes. Remove from heat and stir in candied fruit. Set aside.
2Cream together butter and sugar. Blend in eggs, one at a time. Sift together flour, soda and spices; add to butter mixture alternately with orange juice. Add molasses-fruit mixture and chopped nuts; blend.
3Line mini cupcake pan with liners. Spoon in fruit cake mixture. If desired, garnish with pecan half or an additional cherry half. Bake at 325F for 15-20 min. (check) Cool before storing. Makes 6 doz fruit cake bon bons.
About this Recipe
Course/Dish: Fruit Desserts, Candies
Other Tag: Quick & Easy


text:
Type: Posts; User: schmack
I was half chosen
I am your long lost brother. I found u
Hello guys. Whatsup
I'm your long rlost brother.
herro there
thought you gave up your citizenship?
Why so salty?
But dat bush was wack back then.
erro, lol
so buff
I was rejected as well for failing hearing test on my left ear by 5%. Tried to get a waiver and still didn't fly.
It was for the US Marines.
Good luck OP
I see you deleted the CD. LOL
Too many Emo's on the show. Terrible acting.
Guy that owns the boat should just kill them all.
morning brahs
Oh hell no.
He's lucky it didn't grab the kids arm when he reached out.
The bad thing about Florida is no decent hikes. At least none that I am aware of.
thanks for the info. Yeah I saw the pics but also saw the foot traffic on the sandy ground so wasn't sure. It's good to hear what you mentioned and I will plan a time to make it there. I also don't...
Thanks Barbrox I was thinking that's the case. Similiar to New England White Mountains
I see that place all over social media now. Can you tell me if it felt very commercialized or crowded when you went?
Anyone know the conditions up in Washington State for hiking in the end of May?
Love Red Robin, afraid of Sonic since it's never busy.
How is Shake and Steak? Or Steak and Shake? LOL
Chik Fila is nomnom. Went to one nearby and the Xmas lights and decoration was insane....


text:
A Guest Blog by Assistant Principal Lianne Carter (LISTD)
At Turning Pointe School of Dance we offer professional Dance training in Ballet, Tap, Modern and Jazz for children aged 18 months all the way up to Advanced Level, including adult classes. We are a well-established school with a 50 year history in the area, and now offer classes in Haywards Heath, Lindfield, Plumpton, Barcombe and Lewes.
We value every pupil as an individual, and whilst many of our students are offered places at prestigious conservatoires and universities (including The Royal Ballet School, London Studio Centre, The BRIT School, London Contemporary School of Dance, Trinity Laban, and Northern School of Contemporary Dance) others attend lessons purely as a hobby and a form of exercise, but the escapism and enjoyment they gain from the classes is just as important.
Our pupils have the opportunity to take exams in RAD Ballet and ISTD Modern and Tap if they wish to do so, and every two years we put on a large production in a professional theatre. We also enjoy performing at community events like Lindfield Arts Festival, and hope that you will enjoy watching our dancers!
We have recently launched Melody Bear Pre-School Dance classes and adult Ballet-Fit classes on Friday mornings at Clair Hall. If you would like further information on any of our classes please email email@example.com. Please also take a look at our website- www.turningpointedanceschool.co.uk


text:
|Home | About | Journals | Submit | Contact Us | Français
Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate–binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements.
Numerous organelles undergo membrane fission and fusion events during cell division or vesicular traffic or in response to changes in environmental conditions. Examples include the Golgi (Acharya et al., 1998 ), mitochondria (Bleazard et al., 1999 ), peroxisomes (Kuravi et al., 2006 ), and lysosomes (Ward et al., 1997 ). In the yeast Saccharomyces cerevisiae, the vacuole is the terminal component of the endocytic pathway. It serves as a lysosome-like hydrolytic compartment and as a major storage compartment and plays an important role in osmoregulation and ion homeostasis. Owing to its large size, this organelle represents a very good object with which to study membrane dynamics in vivo and in vitro (Kane, 2007 ).
On budding of yeast cells, the vacuole of the mother cell pinches off vesicles that migrate into the daughter cell, where they fuse to form a new vacuole (Weisman, 2003 ). Vacuolar morphology also changes in response to environmental conditions such as nutrient availability or the osmolarity of the medium. When exposed to hypertonic conditions, yeast cells divide their large vacuoles into numerous smaller ones, probably in order to readapt the surface-to-volume ratio of the compartment after the osmotic loss of water. The dynamin-like GTPase Vps1p is found on vacuoles (Peters et al., 2004 ) and is implicated in the regulation of vacuolar fission and fusion. It bears the three classic dynamin domains, including a GTPase domain, a middle domain, and a GTPase effector domain (Rothman et al., 1990 ; Vater et al., 1992 ; Yu and Cai, 2004 ), but, unlike classic dynamins, it does not contain a PH domain. Dynamins can generate force and support many membrane fission processes (Hinshaw, 2000 ; Praefcke and McMahon, 2004 ). Vacuole fission also requires the proton pump activity of the vacuolar H+-ATPase (Baars et al., 2007 ) and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2; Gary et al., 1998 ; Bonangelino et al., 2002 ). The concentration of PI(3,5)P2 increases drastically upon osmotic stress. PI(3,5)P2 in yeast is synthesized from phosphatidylinositol by two sequential phosphorylations by the phosphoinositide 3-kinase (PI 3-kinase) Vps34p and the phosphatidylinositol 3-phosphate (PI(3)P) 5-kinase Fab1p (Cooke et al., 1998 ; Gary et al., 1998 ). Fab1p carries a FYVE (Fab1, YOTB, Vac1, EEA1) domain that binds PI(3)P and localizes to the vacuole (Efe et al., 2007 ). Fab1p kinase activity is regulated by three other proteins: Vac7p, Vac14p, and Fig4p (Bonangelino et al., 2002 ; Dove et al., 2002 ; Gary et al., 2002 ). Vac7p and Vac14p were discovered in screens for mutants defective in vacuole inheritance (Gomes de Mesquita et al., 1996 ; Wang et al., 1996 ). They activate Fab1p. Fig4p dephosphorylates PI(3,5)P2 to PI(3)P. Together with Vac14p, Fig4p is also necessary for Fab1 complex assembly (Gary et al., 2002 ; Botelho et al., 2008 ; Jin et al., 2008 ), which could explain its second role in PI(3,5)P2 synthesis. Deletion of VAC7, VAC14, or FAB1 leads to an enlarged vacuolar phenotype (Weisman, 2003 ). A link of the Fab1 complex to organelle transport into the bud during cell division is provided by Atg18p, a PI(3)P- and PI(3,5)P2-binding protein that regulates Fab1 activity and binds Vac17p, an adaptor protein mediating the attachment of vacuoles to myosin V and actin (Tang et al., 2003 ; Efe et al., 2007 ).
Two additional proteins involved in vacuole fission are the CORVET subunit Vps3p (LaGrassa and Ungermann, 2005 ; Peplowska et al., 2007 ) and the kinase Yck3p. Vacuoles are retained in the fragmented state by inhibiting fusion through a Yck3p-dependent phosphorylation of a subunit of the Rab-effector homotypic fusion and vacuole protein sorting complex, Vps41p (Cabrera et al., 2009 ). Recently vacuole fragmentation has also been linked to nutritional conditions through the target of rapamycin complex (Michaillat et al., 2012 ).
Vacuole fusion and fission change the surface-to-volume ratio of the organelle. Such adjustments are probably required upon changes in osmotic value, upon starvation, when the vacuolar hydrolytic enzymes are massively up-regulated, or for storage of reserve compounds, such as amino acids or polyphosphates (Kane, 2007 ). Here we used hypertonic stress to modify vacuole structure because it allows us to induce rapid, synchronous, and homogeneous vacuole fragmentation in a yeast population. This allowed us to study the process in real time.
Different scenarios can be invoked to explain how the vacuolar surface-to-volume ratio might be adapted to hypertonic stress (Figure 1A). It is unknown whether vacuoles divide equally in the middle, providing two fragmentation products of similar size, which might then undergo further rounds of fragmentation in order to produce the small vacuolar vesicles that are the final products of osmotically induced vacuole fragmentation. Alternatively, the organelle might fragment asymmetrically, producing small vesicles from a large central vacuole by a mechanism similar to budding. We address this question by time-resolved confocal microscopy of living yeast cells.
Yeast vacuoles can be stained in living cells with the lipophilic dye FM4-64 (Vida and Emr, 1995 ). FM4-64–stained cells were immobilized on concanavalin A–coated chambered cover slides filled with medium. This attachment does not prevent normal growth and division of the cells (Supplemental Movie S1). After the start of image acquisition, vacuole fragmentation was induced on the microscope stage by supplementing the cell suspension with 0.5 M NaCl. Already seconds after this addition, the vacuoles shrank and began to form invaginations (Figure 2, A and B), leaving heteromorphic protrusions in their vicinity. The invaginations are distinct from vacuolar invaginations involved in micronucleophagy at nuclear–vacuolar junctions since neither the nuclear vacuolar junction protein Nvj1p (Pan et al., 2000 ) nor the nucleolar marker Nop1p (Henriquez et al., 1990 ; Dawaliby and Mayer, 2010 ) localized to them, and a strain deleted for NVJ1 is proficient for vacuole fragmentation. They are also unrelated to microautophagic invaginations because they could be observed in mutants that impair microautophagy, such as Δatg18 (see later discussion), Δatg5, and Δvtc4 (Muller et al., 2000 ; Sattler and Mayer, 2000 ; Uttenweiler et al., 2007 ).
The initial invagination phase, which lasted <1 min, was followed by a continuous remodeling of the vacuole. In this phase, the heteromorphic protrusions that were left in the vicinity of the invaginations rounded off and produced small, spherical structures that stayed attached to the remaining large central vacuole. This phase took 10–15 min to complete. After 15 min, all wild-type cells exhibited numerous spherical structures (Figure 2C). We preferentially use the background BJ3505 because it provides big cells and vacuoles that are well suited for light-microscopic analysis. Furthermore, its pep4 mutation reduces proteolytic artefacts in biochemical fractionations, which will be performed in future analyses of the fragmentation reaction. Cells from other strain backgrounds behaved similarly as BJ3505 (Figure 2D), indicating that the mode of vacuole remodeling is not strain specific. To confirm our observations from light microscopy, we recorded electron micrographs of yeast cells at different times after osmotic shock (Figure 2E). At 45 s past the salt shock, most vacuoles exhibited invaginations of various sizes, similar to what was seen before by fluorescence microscopy. After 2 min, cells with smaller spherical structures became more numerous, and after 5 min, few invaginations remained, and small, spherical structures predominated. At the 15-min time point, all cells showed exclusively small round vacuolar structures.
Next we tested whether the spherical structures that appeared were products of true vacuole fragmentation, that is, vacuolar vesicles separated from the rest of the organelle. They might as well represent vacuolar invaginations or evaginations that were optically sectioned, a process that would also yield circular profiles. In addition, lumenal vesicles that pinched off into the interior of the vacuole could result in similar images in the light microscope. Owing to the limited resolution of the confocal microscope, we could not unequivocally address this problem by three-dimensional reconstruction. Therefore we used a strain expressing a cytosolic version of green fluorescent protein (GFP), which would fill any invagination of the vacuolar membrane and also lumenal vesicles generated from it. We found examples for clearly identifiable vacuolar invaginations that were longitudinally sectioned and accordingly colored by GFP, confirming the validity of the approach. The large majority of spherical structures generated from the invaginated vacuoles, however, did not show lumenal GFP staining (Figure 3A). Therefore these spherical structures retained the outside-out configuration of the vacuolar membrane. That these newly formed structures were completely detached from the original vacuole could be demonstrated by fluorescence recovery after photobleaching (FRAP) analysis. We used a strain expressing a GFP fusion of the membrane-integral vacuolar V-ATPase subunit Vph1p (Figure 3, B and C). If one of the vesicles from a freshly fragmented cluster of vacuoles was bleached with a laser, its fluorescence signal did not recover by delivery of protein from the other vesicles in vicinity. Correspondingly, the fluorescence signal of these neighboring vacuoles was retained during the laser pulse, as well as after the photobleaching. We only observed a slow, constant bleaching that was common to all fluorescent structures and due to continued image acquisition. These data suggest that the spherical structures formed after the osmotic challenge represent vacuolar vesicles completely separated from each other.
We measured the surface and volume of the vacuoles before and after the reaction. Their volume decreased by 75% (Figure 3D), whereas the apparent surface area remained almost constant and decreased only by 20%. The apparent loss of surface might be due to the limited resolution of the light microscope, which precludes resolution of small tubular or vesicular membrane structures adjacent to a vacuolar boundary membrane. In addition, membrane shriveling (Figure 1B) can reduce the apparent vacuole surface. We could observe vacuole shriveling in a low percentage of cells and suppose that it might remain undetectable in the majority of the cases if it is not sufficiently pronounced.
The proton pump activity of the V-ATPase and the resulting proton gradient across the vacuolar membrane are required for vacuole fragmentation (Baars et al., 2007 ). We used a Δvma1 strain, which lacks a catalytic subunit of the V-ATPase, in order to test which step of vacuole fragmentation depends on the vacuolar proton gradient. Δvma1 cells showed a single large, round vacuole (Figure 4A). On hypertonic shock, vacuoles from Δvma1 cells did not show any deep invaginations at the 2-min time point, when they are highly prominent in wild-type cells (Figure 2A). The vacuoles showed only a slight shrinking and shriveling of the vacuolar membrane, leading to shallow indentations that rapidly disappeared during the next 10–15 min without producing vacuolar fragments. Even those shallow invaginations were much less frequent than the pronounced invaginations in the wild type. Owing to the vacuolar acidification defect these mutants exhibit weaker FM4-64 staining of the vacuolar boundary membrane and an enhanced lumenal background staining, probably reflecting the intravacuolar accumulation of multivesicular body (MVB) vesicles (Wurmser and Emr, 1998 ). We also tested the effect of pharmacological suppression of V-ATPase function in wild-type cells. This more acute treatment can circumvent secondary effects resulting from the constitutive absence of V-ATPase activity in deletion mutants (Kane, 2007 ). In addition, short treatment of wild-type cells with a potent inhibitor of the vacuolar H+-ATPase, concanamycin A (Drose and Altendorf, 1997 ), blocked deep vacuolar invagination after salt shock and permitted only shallow, less frequent indentations (Figure 4B). Quantification over time illustrates this fact (Figure 4C). This suggests that the electrochemical potential over the vacuolar membrane is necessary for the deep vacuolar invaginations that precede fragmentation. In line with this, inactivation of V-ATPase also impedes the reduction of vacuolar volume upon hypertonic shock (Figure 3D).
The dynamin-like GTPase Vps1 is necessary for both vacuole fragmentation and fusion in yeast (Peters et al., 2004 ; Michaillat et al., 2012 ). We tested which phase of vacuole fragmentation was affected by this protein. Cells from a Δvps1 deletion strain show a large, round central vacuole surrounded by smaller vesicles. When Δvps1 cells were exposed to a salt shock, their large, round vacuoles did not fragment (Figure 5, A and B) and showed reduced shrinking. Their invaginations were much shallower and less numerous than those in wild-type cells (Figure 5, A–C). They formed more slowly, with a half-time of 20 instead of 10 s for the wild type. They were also unstable and disappeared within a few minutes (Figure 5D). Δvps1 vacuoles did not produce normal-sized vacuolar fragmentation products from their large central vacuoles upon salt treatment, but they showed additional, poorly resolvable tubulovesicular evaginations emanating from the surface of the large central vacuole. These data suggest that Vps1p already influences the invagination of the vacuolar membrane. This early defect interferes with attempts to assay a contribution of Vps1p to the subsequent scission of vacuolar fragments, which we nevertheless expect to exist, due to the well-characterized fission activities of dynamin-like GTPases (Schmid and Frolov, 2011 ).
The level of PI(3,5)P2 increases up to 20-fold upon osmotic stress, and PI(3,5)P2 regulates vacuolar morphology. PI(3,5)P2 is produced by a protein complex of the catalytic subunit Fab1p and its regulatory subunits Vac7p, Vac14p, and Fig4p. Cells deleted for the PI(3,5)P2-producing kinase Fab1 show single enlarged vacuoles and are defective in vacuole inheritance and vacuole fragmentation (Yamamoto et al., 1995 ; Wang et al., 1996 ; Dove et al., 1997 ; Cooke et al., 1998 ; Gary et al., 1998 ; Bonangelino et al., 2002 ; Jin et al., 2008 ). On a salt shock, vacuoles of Δfab1 cells still formed deep invaginations at a high frequency, but they could not form vacuolar fragments (Figure 6, A and B). Unlike the labile invaginations in Δvps1 cells, the invaginations in Δfab1 cells persisted for the entire observation period of 15 min (Figure 6E). After prolonged incubation, the initial invaginations rounded up and formed spherical structures in the interior of the vacuole. These structures contain engulfed cytosolic material, as demonstrated by their staining with cytosolic fluorescent probes such as soluble GFP or FYVE2-GFP (see later discussion). They were mobile inside the vacuoles, suggesting that they had detached from the boundary membrane. Similarly, cells lacking the Fab1p activator Vac7p, which are also defective for vacuole fragmentation (Gary et al., 1998 , 2002 ), showed long-lived invaginations, but intravacuolar spherical structures were less frequent (Figure 6C). In addition, a Δvac14 mutant (Bonangelino et al., 2002 ; Dove et al., 2002 ; Jin et al., 2008 ) showed a qualitatively similar defect in the formation of vacuolar fragments, which was, however, less pronounced than in Δfab1 cells (Figure 6D). The less pronounced effects of the noncatalytic subunits of the Fab1 complex are likely due to the persistence of small amounts of PI(3,5)P2 in these strains (Efe et al., 2007 ).
We also analyzed cells lacking the PI 3-kinase Vps34p (Schu et al., 1993 ), which produces the substrate for Fab1p. Vps34p exists in two PI 3-kinase complexes—an autophagosomal complex I and the endosomal/vacuolar complex II (Kihara et al., 2001 ; Burda et al., 2002 ). The vacuoles in Δvps34 cells did not fragment (Figure 7A). Deletion of the gene for the endosomal/vacuolar complex II subunit Vps38p (Figure 7C) significantly reduced salt-induced vacuole fragmentation, whereas deletion of the gene for the autophagosomal complex I subunit Atg14p (Tsukada and Ohsumi, 1993 ; Kametaka et al., 1998 ; Kihara et al., 2001 ) had no effect (Figure 7D). Closer inspection of the fragmentation process revealed that Δvps34 cells showed pronounced vacuolar invaginations upon salt treatment. Although the vacuoles in both Δvps34 and Δfab1 cells did not fragment, the invaginations in Δvps34 decayed during the 15 min of observation, whereas in Δfab1 cells they remained stable. Δfab1 cells not only fail to produce PI(3,5)P2 but also accumulate increased levels of PI(3)P, suggesting that accumulating PI(3)P might stabilize vacuolar invaginations and that its metabolization into PI(3,5)P2 might be needed to vesiculate the membrane.
This hypothesis is consistent with results from our attempts to localize PI(3)P. Membranes containing PI(3)P can be labeled in living cells with a probe containing two PI(3)P-binding FYVE domains from the human Hrs protein fused to GFP (Gillooly et al., 2000 ). Expression of this probe in Δfab1 cells brightly stains foci on the vacuolar boundary membrane and vacuolar invaginations (Figure 8A, arrowheads). As invaginations form during fragmentation, those foci move to invaginated regions and concentrate there. Wild-type cells also show FYVE2-GFP foci on the vacuolar boundary membrane and in invaginated regions upon salt addition. In contrast to the persistent signal on the intravacuolar structures in Δfab1 cells, however, the foci in wild-type cells dissociated again in the course of fragmentation (Figure 8B). This suggests a transient enrichment of PI(3)P on invaginating regions of the vacuoles during fragmentation.
In search of potential effectors of PI(3)P and PI(3,5)P2, we tested proteins known to bind these lipids. Atg18p is a vacuole-associated protein that binds PI(3,5)P2 with high affinity and negatively regulates PI(3,5)P2 production (Dove et al., 2004 , 2009 ; Stromhaug et al., 2004 ; Efe et al., 2007 ). Cells lacking Atg18p show a drastically increased steady-state level of PI(3,5)P2 and enlarged vacuoles. We observed that in Δatg18 cells vacuole fragmentation is significantly delayed (Figure 9, A and B). Δatg18 differs from other mutants affecting the Fab1 complex in that it displays enlarged vacuoles and vacuolar fragmentation problems despite the fact that it shows no reduction in PI(3,5)P2 at the whole-cell level. Therefore we tested whether Fab1p might be mislocalized in a Δatg18 cell, which might allow synthesis of PI(3,5)P2 but not in the place where it is required. We generated cells expressing a Fab1p-GFP fusion as the sole source of Fab1, either in the presence or absence of ATG18. In both cases, Fab1p-GFP showed the same localization to the vacuolar rim. It was concentrated in an inhomogeneous manner on the vacuoles, confirming earlier observations (Bonangelino et al., 2002 ).
On hypertonic treatment, vacuoles shrink within seconds, probably to compensate for the water efflux from the cytosol to the surrounding medium. Shrinking is accompanied by tubular invaginations of the vacuole. Vesicles are formed from the finger-like protrusions remaining between them. These observations raise several interesting questions. First, why do vacuoles fragment at all in an active, protein- and lipid-dependent manner? It appears that many vacuolar functions, such as hydrolytic degradation or the storage of polyphosphates, amino acids, and polyamines, might also work in a shrunken organelle that is not round. A major difference between a deflated and an inflated state of an organelle is the tension of its membrane. Shrinking changes the surface-to-volume ratio and eliminates membrane tension. Fragmenting the organelle into multiple smaller copies readjusts the surface-to-volume ratio and hence allows reestablishment of tension of the vacuolar boundary membrane. Membrane tension can influence the activity of channel and transport proteins (Hamill and Martinac, 2001 ). Vacuoles contain numerous channels and transporters, which are crucial for its function in storage and release of various compounds. Some of them might be affected by membrane tension, for example, the vacuolar Ca2+ channel Yvc1p, which releases Ca2+ upon hypertonic shock (Chang et al., 2010 ). In addition, membrane tension may be necessary to allow vesicular traffic to the organelle. Fusion between vacuoles and vesicular transport to them depend on the Rab-GTPase Ypt7p (Wada et al., 1992 ; Wichmann et al., 1992 ; Haas et al., 1995 ; Mayer and Wickner, 1997 ), and the function of Ypt7p is influenced by membrane tension (Brett and Merz, 2008 ). Thus it is likely that vacuolar membrane tension needs to be maintained to sustain vacuolar membrane trafficking routes.
A second interesting aspect is the fact that fragmentation happens asymmetrically. It immediately produces fragmentation products of the final size rather than proceeding through a series of equal divisions to generate vesicles of increasingly smaller size (Figure 1). Separating small vesicles with a high surface-to-volume ratio should permit more rapid readjustment of this ratio and the regaining of functionality of the compartment because already the first fragmentation products will possess a drastically increased surface-to-volume ratio.
A third interesting aspect is the involvement of the different fragmentation factors at different phases of the process (Figure 10). Osmotically induced invaginations of the vacuolar membrane might be taken as a passive shape change dictated by the efflux of water and loss of volume, but this seems not to be the case. Invagination can be suppressed by deletion of either the V-ATPase or the dynamin-like GTPase Vps1p. Salt stress stimulates rapid assembly of the V1 and V0 sectors of the V-ATPase (Li et al., 2012 ). The resulting augmented electrochemical gradient across the vacuolar membrane might directly affect distribution and properties of vacuolar lipids in order to support its large-scale deformations. Changes in the electrochemical membrane potential can directly induce transbilayer lipid asymmetry (Farge and Devaux, 1992 ; Mui et al., 1995 ; Sackmann and Feder, 1995 ) and lateral phase separations of lipids (Schaffer and Thiele, 2004 ). Such changes are sufficient not only to tubulate pure two-phase lipid systems, but also to allow vesicle scission from them (Julicher and Lipowsky, 1993 ; Lipowsky, 1995 ).
A deficiency in proton pumping could also affect the vacuolar membrane by influencing the turnover of vacuolar contents. The major vacuolar compounds are polyphosphates, which are synthesized by the vacuolar VTC complex (Hothorn et al., 2009 ) and can form up to 30% of the dry weight of yeast (Liss and Langen, 1962 ). Polyphosphates influence vacuolar membrane dynamics, as illustrated by their roles in vacuolar invagination during microautophagy (Uttenweiler et al., 2007 ) and in vacuole fusion (Muller et al., 2002 , 2003 ). Their turnover depends on an endopolyphosphatase that must be matured by vacuolar hydrolases, a process that probably depends on vacuolar acidification (Sethuraman et al., 2001 ; Shi and Kornberg, 2005 ). Polyphosphates contain up to hundreds of phosphate residues, are highly negatively charged, and form complexes with Ca2+, Mn2+, Mg2+, basic amino acids, and other monovalent cations (Rao et al., 2009 ). It is conceivable that polyphosphates might also associate with and cluster charged lipids, either by direct binding or by ionic bridges via bivalent cations. Because uptake and turnover of most vacuolar compounds depend on H+-driven transporters (Kane, 2007 ), perturbation of the proton gradient could interfere with vacuolar invagination by affecting vacuolar ion balance and lipid distribution.
We observed an unexpected early role for Vps1p in fragmentation since Δvps1 vacuoles do not show the large invaginations that can be observed in wild-type cells. The membrane in invaginated areas is negatively curved, but dynamin-like proteins bind to membrane areas of high positive curvature and can thereby promote tubulation and scission of membranes (Roux et al., 2010 ; Schmid and Frolov, 2011 ). If the role of Vps1p for forming the invagination was related to its binding to positively curved regions, it could only affect the rim of a forming indentation of the vacuolar boundary membrane. Here the membrane is positively curved. Vps1p might thus stabilize the rims of the invaginating structures. In this way, Vps1p should also be enriched at the tips of the remaining finger-like structures that can be observed between invaginations, that is, at the sites where scission of the final fragmentation products occurs. We could not test this model directly by microscopy because we were not able to produce tagged versions of Vps1p that showed a normal invagination pattern, although our tagged versions were functional for other aspects of Vps1p activity, such as endocytosis or vacuole fusion (Peters et al., 2004 ; Smaczynska-de Rooij et al., 2010 ). Attempts to localize Vps1p by immuno–electron microscopy have not succeeded. Our observation of a role of Vps1 in the formation of invaginations is consistent with observations of Hyams and coworkers in Schizosaccharomyces pombe, who ascribed to Vps1p a function in tubulating vacuoles (Rothlisberger et al., 2009 ). In S. pombe, vacuole scission required an additional dynamin-like GTPase, Dnm1p. In S. cerevisiae, however, we observed that vacuole fragmentation in a Δdnm1 mutant occurs normally (unpublished data).
The locally appearing tubules are probably accompanied by changes in the lipid phase in those areas. Our study illustrates this for one lipid, PI(3)P. On hypertonic shock, the amounts of PI(3,5)P2 on the vacuole increases 10- to 20-fold (Dove et al., 1997 ; Bonangelino et al., 2002 ). In addition, the levels of PI(3)P rise, although more moderately. Live-cell imaging of a strain deleted for the PI(3)P 5-kinase Fab1p shows that the mutant vacuoles invaginate even more vigorously than those of wild-type cells, whereas the actual formation of new vesicles is drastically reduced and delayed. Instead, the deep invaginations evolve into spherical structures that accumulate inside the vacuole. We consider those as degenerated or "frustrated" invaginations. They show a high level of PI(3)P. Because cells lacking Fab1p accumulate PI(3)P, these spherical invaginated structures may result from the hyperaccumulation of PI(3)P due to the inability to convert it into PI(3,5)P2. In line with this, a Δvps34 strain that no longer produces PI(3)P does not show this increased invagination activity and does not accumulate intravacuolar spherical structures. We hypothesize that PI(3)P and PI(3,5)P2 could act sequentially in vacuole fragmentation. PI(3)P, produced from PI 3-kinase complex II, might stabilize invaginations, and its conversion to PI(3,5)P2 might induce the subsequent fission of vesicles from the membrane protrusions remaining between the invaginations. A surplus in PI(3)P might recruit proteins that induce negative curvature and stabilize the invaginations, eventually leading to the observed spherical structures if PI(3)P is not converted into PI(3,5)P2.
The formation of PI(3)P and PI(3,5)P2 from PI might itself influence membrane curvature, but the change in the head group is rather small. We consider it as more likely that these lipids operate by recruiting lipid-binding proteins, which then help to shape the membranes. A candidate for such a factor is Atg18p, a PI(3,5)P2-binding protein that regulates Fab1p activity (Dove et al., 2004 ; Efe et al., 2007 ). Atg18p is recruited to the vacuolar membrane after hypertonic shock. Δatg18 cells fragment their vacuoles less well than wild-type cells, although they have even more PI(3,5)P2. In other mutants affecting the Fab1 complex the situation is inverse, that is, their fragmentation defects correlate to strong reductions in PI(3,5)P2 levels. The fragmentation defect of Δatg18 cells might result from the perturbations caused by the increased PI(3,5)P2 level. This, however, seems unlikely because fab1-5 mutants, which show a similar increase in PI(3,5)P2 as Δatg18 cells, have hyperfragmented vacuoles (Gary et al., 2002 ; Efe et al., 2007 ). Thus it is more likely that Atg18p supports the transition from invaginated to fragmented vacuoles independent of its influence on the conversion of PI(3)P to PI(3,5)P2, perhaps via its interaction with PI(3,5)P2 and resulting influences on membrane curvature.
Fragmentation of vacuoles happens not only during adaptation to changes in the osmotic environment of the yeast, but also during the cell cycle. The vacuole in the mother cell forms an elongated structure, which extends into the bud and can pinch off tubulovesicular structures (Weisman, 2003 ). When the bud neck closes, driven by the septins and an actin–myosin ring, these structures are separated from the mother vacuoles, where they fuse again to form the vacuole of the daughter cell (Weisman, 2003 ). Lack of Fab1p delays this process, whereas cells lacking Vps1p or a functional V-ATPase appear not to be deficient for vacuole inheritance (unpublished observation). The independence of vacuole inheritance from two factors implicated in salt-induced fragmentation suggests that the rather slow fragmentation during cell division may not require all of the factors necessary for the fast adaptation to hyperosmotic shock. Inversely, there are factors necessary for vacuole inheritance that do not influence osmotically induced vacuole fragmentation. In vacuole inheritance, a major force-providing factor for the formation of the thin segregation structures growing out of the vacuole and their migration toward the bud is the myosin-driven transport of vacuoles along actin cables (Hill et al., 1996 ; Catlett and Weisman, 1998 ). This factor most likely does not play an active role during osmolarity-induced fragmentation, since we observed that this process is insensitive to the actin depolymerizing drug latrunculin B, as well as to various mutations interfering with actin function (unpublished data).
Careful examination of the morphological changes of the vacuole during salt-induced fragmentation allowed us to dissect the process into two distinct phases with nonoverlapping requirements for the known fragmentation factors. This dissection and the fact that vesiculation happens in an asymmetrical manner at sites that are identifiable in the light microscope provides an important tool for future identification of additional proteins involved in vacuole fragmentation and for studying how they shape the membrane.
Culture media were either normal yeast extract/peptone/dextrose (YPD; 2% glucose) or YPD buffered to pH 5.5 (for Δvma strains). Cultures were shaken at 30°C and 150 rpm. Strains carrying expression plasmids were grown on the corresponding Hartwell's Complete dropout medium. Strains used in this study are listed in Table 1. Deletion mutants were generated by replacing the gene with either a loxP-kanMX-loxP cassette from plasmid pUG6 (Guldener et al., 1996 ) or a nourseothricin (clonNat) cassette from plasmid pFA6a-natNT2 (Janke et al., 2004 ). Primers used for amplification of those cassettes are as follows: Δfab1, 5′-tcg aat agc aag gta gct tcc ATC CTG TAC ATG CAA GAC CCG TAC GCT GCA GGT CGA C-3′ and 5′-ACC ACG GAT CAG GAA CCA TCA AAA TAT ACC TCT CCA TTG CAT CGA TGA ATT CGA GCT CG-3′ ; Δvac7, 5′-GTA GTA GCA CCT AAT CCT TCT ATT CCC TCT GCC TCC ACA TCC GTA CGC TGC AGG TCG AC-3′ and 5′-CTG GAA TAA ACT CAT CGT GAA GGT TAG TGT GTT GCG GTC GAT CGA TGA ATT CGA GCT CG-3′; Δvac14, 5′-GGT CAA ACA ATG CGT TCT AGA AGG GGA CTA TGA TCG TAT TGC GTA CGC TGC AGG TCG AC-3′ and 5′-CTT TGG CTA ACG GCA CTT TGC GAG ATA TCA GAA TTG GAA TCA TCG ATG AAT TCG AGC TCG-3′; and Δatg18, 5′-ATA GTG TTC CAG TTA ACT CTG TAT CCT TTT CTC TTC GGC CTG ACA CAG CTG AAG CTT CGT ACG C-3′ and 5′-TGC GTT GTG ACG TAC GGA AGG CAG CGC GAG ACA CTT CCG TGA TCA GCA TAG GCC ACT AGT GGA TCT G-3′.
The pGEX vector for expression of the glutathione S-transferase–tagged double-FYVE finger from mammalian Hrs (Gillooly et al., 2000 ) was cut with EcoRI and SalI and the excised FYVE2-sequence ligated in a pUG36:eGFP vector under the control of a MET25 promoter, resulting in expression of eGFP-FYVE2. The VPH1-GFP plasmid expressing VPH1 under its own promoter has been described previously (Dawaliby and Mayer, 2010 ), as has the GFP-PHO8 plasmid expressing PHO8 under its endogenous promoter (Baars et al., 2007 ).
Cells were inoculated from a preculture in stationary phase and grown overnight to logarithmic phase (OD600 between 0.2 and 0.8). After dilution to OD600 of 0.2 in 2-ml culture, FM4-64 (10 mM in dimethyl sulfoxide [DMSO]) was added to a final concentration of 25 μM. Cells were stained for 1 h, followed by three washing steps (2 min, 3000 × g) and a subsequent chase of 1–2 h, depending on the endocytotic efficacy of the strain.
Concanavalin A from an aqueous 10 mg/ml stock solution was diluted 10-fold with water. A 35-μl portion was spotted onto LabTek eight-well chambered cover slides and air dried. After the chase period, yeast cells were centrifuged at 2000 × g for 3 min and resuspended in 50 μl of fresh medium. A 25-μl portion of the cell suspension were spotted on the previously coated slides and incubated for 5 min. After two washing steps with 400 μl of fresh medium, the cells were kept in 200 μl of medium for imaging. Microscopy was performed at room temperature (22°C). An equal volume of medium containing 1 M NaCl was added during visualization to induce the salt shock. Pictures were taken with an UltraView Vox confocal spinning disk unit (PerkinElmer-Cetus, Waltham, MA) connected to an inverted Zeiss microscope (Carl Zeiss, Jena, Germany) with a 100× oil immersion objective with a numerical aperture of 1.41 and a Hamamatsu C9100-50 camera (Hamamatsu, Hamamatsu, Japan). For colocalization with FM4-64, GFP was excited at 488 nm and imaged using a 527/55-nm bandpass filter. FM4-64 was excited at 488 or at 561 nm for colocalization, respectively. For FRAP analysis, we used the Photokinesis unit of the UltraView Vox system and applied one cycle of 200 ms with full laser intensity at 488 nm to bleach GFP in the target section. Pictures were contrast enhanced with ImageJ (National Institutes of Health, Bethesda, MD) and Photoshop (Adobe, San, Jose, CA). In general, image stacks were recorded at z-distances of 200–500 nm in order to cover the entire depth of the cells. Maximum-intensity projections of the stacks were produced with the ImageJ software and used to evaluate the fragmentation process. The scale bar on all fluorescence pictures corresponds to 5 m.
For quantification of the percentage of cells belonging to three different fragmentation stages, full chips from three independently recorded image series were counted (30–60 cells per picture), resulting in a total of 100–200 cells per strain. The percentage of every class was calculated for every image series, and the mean values and standard deviations were determined from those values. For calculating the surface-to-volume ratio, a total of 15 vacuoles from thee different series were analyzed. Vacuoles were assumed to be spheres. Their diameters in the xy plane were measured at least in three different orientations, averaged, and used for calculation.
Cells were stained with FM4-64 as described. Concanamycin A was added to the cells at the beginning of the chase period and maintained in all washing steps and on the chambered cover slide. The final concentration of concanamycin A was 2 μM, added from a 200 μM stock in DMSO.
Cells were grown in YPD to logarithmic phase. In the morning, they were diluted to OD600 nm of 0.2 in fresh YPD supplemented with 1% gelatin to improve embedding. Hyperosmotic stress was induced at room temperature by mixing 0.5 ml of cell culture with 0.5 ml of YPD supplemented with 1% gelatin and 1 M NaCl. At different time points after hyperosmotic stress, samples were concentrated by centrifugation (10 s, 2000 × g, room temperature), and the pellets were immediately transferred to planchettes of 200-μm depth, cryoimmobilized with HPM 010 high-pressure freezer (Bal-Tec; Leica Microsystems, Buffalo Grove, IL), and stored in liquid nitrogen. Freeze substitution of the samples was carried out in an automated freeze substitution device (Leica Microsystems) in methanol containing 1% OsO4 (Merck, Darmstadt, Germany) for 24 h at −90°C. Then samples were warmed (5°C/h) to −30°C (3 h) and finally warmed up to 0°C before removal of the substitution medium and embedding in Epon. Contrasted ultrathin sections (70 nm) were observed in a Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands) operated at 120 keV. Images were taken on an Eagle 4k × 4k camera (FEI) with TIA acquisition software.
We thank Yoshinori Ohsumi, Christian Ungermann, and Margarita Cabrera for strains. We are grateful to Véronique Comte, Monique Reinhardt, and Andrea Schmidt for providing valuable technical assistance and to the Electron Microscopy facility of the University of Lausanne for help in electron microscopy. This work was supported by grants from the SNF (Schweizerischer Nationalfonds) and the ERC (European Research Council) to A.M.
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-05-0347) on July 11, 2012.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
An "alternate" climate summit organised by the well-known once-hippie community of Christiania here ran into trouble early on Tuesday when activists clashed with police, leading to 210 arrests. Police used dogs, batons, teargas, flash bombs and water cannons to disperse the activists.
The clash occurred at an overnight debate organised by the NGO Climate Justice Action. The organisers claimed that around midnight, police surrounded a bar where the debate was taking place, and set off teargas canisters all around.
Some of the speakers had been sharply critical of the Dec 7-18 UN climate summit going on here. Many NGOs have been attending the 'alternate' summit instead, saying people had to tackle climate change on their own because governments were not going to.
Police said five of those arrested were still in custody and had been charged with attacking policemen. Police official added that the authorities intervened when protesters set fire to barricades in front of Christiania, for long a self-proclaimed free zone in the city. Police also claimed to have seized several Molotov cocktails and fireworks during a search.
Denmark introduced special legislation prior to the UN summit allowing police to hold suspects without charge for up to 12 hours.
Long-time Christiania resident Oskar Dresling, who was listening to the debate, said: "There was no reason for this attack. They think that by attacking us while we party, we'll be afraid to come out on the streets this week.
"But we will not be deterred so easily. Climate change is too important an issue to be left to governments that terrorise their own people like this."
The entire Christiania area was cordoned off for over an hour. Richard Bernard of Climate Justice Action said the arrested activists had been forced to squat on the street in the cold.
There had been a clash between police and activists last Saturday as well, leading to over 1,200 arrests, though only nine people had been charged and remained in custody Tuesday.
Christiania is a former army base, which since the 1970s has been a haven for the alternative movement in the Danish capital.


text:
RTE and CTE mRNA export elements synergistically increase expression of unstable, Rev-dependent HIV and SIV mRNAs
© Smulevitch et al; licensee BioMed Central Ltd. 2006
Received: 07 November 2005
Accepted: 13 January 2006
Published: 13 January 2006
Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes.
Posttranscriptional events determine the fate of cellular and viral mRNAs through concerted actions promoting nuclear trafficking and cytoplasmic transport, stabilization and translation. Simian type D (SRV/D) retroviruses and intracisternal A-particle retroelements (IAP) have provided us with unique mRNA transport elements, which utilize conserved cellular export machinery [1–13]. The export of the SRV/D unspliced mRNA is mediated by the cis-acting constitutive transport element CTE [8, 10–13] through interaction with the cellular NXF1 protein [1], which is also the key factor mediating general mRNA export [1–5], a property which is conserved among eukaryotes (reviewed in [14–16]). We previously identified another functionally similar but structurally unrelated posttranscriptional RNA Transport Element RTE [6, 7], which is present in a subgroup of murine IAP. Both CTE and RTE utilize the conserved eukaryotic mRNA transport machinery. Here, we demonstrate that the combination of RTE and CTE in cis leads to synergistic increase in lentiviral gene expression.
Synergistic activation of gene expression in the presence of a combination of RTE-CTE
Synergistic effect of RTEm26CTE on HIV-1 env expression
To rule out that the observed synergistic effect is a unique feature of the gag reporter mRNA, we inserted RTEm26-CTE into an HIV-1 env reporter plasmid pNL1.5E (Figure 2A), expressing the authentic env cDNA from the HIV-1 LTR promoter. Like gag, env is poorly expressed (Figure 2B, lane 8) in the absence of a positive-acting export system, as expected. Both plasmids, containing either CTE (lanes 1, 2) or RTE (lanes 3, 4) alone, showed ~10× fold increase in Env production compared to the pNL1.5E (lane 8). The presence of RTEm26-CTE led to an additional increase in Env production (lane 5). A semi-quantitative analysis using serial dilutions (lanes 5–7) of the cell extract shows a ~100× fold activation, confirming synergistic effect of RTEm26-CTE. This expression level was comparable to that obtained in the presence of Rev (lanes 9–11). These data demonstrate that the synergistic effect of the combination of RTEm26-CTE export elements is applicable for different poorly expressed, unstable HIV-1 mRNAs.
Synergistic effect of RTEm26CTE on expression of a Rev- and RRE-deficient HIV-1 and SIV molecular clones
Upon insertion of CTE or RTE alone into the Rev- and RRE-minus NL4-3, we had previously shown that these RNA transport elements are able to partially replace the viral Rev-RRE system resulting in the production of infectious virus [6, 9, 11, 12] (Figure 3B and 3C). Interestingly, Western immunoblot analysis showed that the presence of RTEm26-CTE mediated a dramatic synergistic increase in expression of both env and gag compared to the clones containing each element alone (Figure 3B). Quantitation of gag expression using an antigen capture assay showed an increase of ~1 log over the presence of CTE or RTE alone. The expression level in the presence of RTEm26-CTE was only slightly lower (~3x-fold) than those obtained by the wild type HIV-1 NL4-3 (Figure 3B). Upon infection of Jurkat cells, the RTEm26-CTE containing Rev-independent HIV-1 clone as well as the RTE- or CTE-containing clones showed similar replicative capacities to levels ~1 log lower than that of the wild type HIV-1 (Figure 3C). Thus, the presence of RTEm26-CTE is able to promote a balanced expression of the viral proteins able to generate infectious virus.
Similarly, we found that the presence of RTEm26-CTE also greatly increased expression of the Rev- and RRE-minus molecular clone of SIVmac239 (Figure 4B) to levels about ~10x-fold higher than those obtained by SIV clone containing only the CTE. Like its HIV counterpart, the RTEm26-CTE-containing SIV produces infectious virus (Figure 4C). We noted that it replicates with growth kinetics similar to the wild type SIV, in both CEMx174 cells (Figure 4C) and monkey PBMCs (data not shown), despite its slightly reduced level of expression (Figure 4B). In contrast to its HIV counterpart, the presence of the more potent RNA export element combination improved the replicative capacity when compared to the virus that contains only the CTE (compare peak at day 14 postinfection). Since we could not test propagation of SIV and HIV in the same cell types, it is possible that cellular factors may contribute to this phenomenon and this was not further investigated.
In conclusion, we have shown that the potent posttranscriptional effect of the RTEm26-CTE combination of RNA export elements from simple expression vectors (Figures 1 and 2) as well as from the complex array of mRNAs produced from the molecular clones of HIV and SIV (Figures 3 and 4).
Synergy depends on the spatial arrangement of RTEm26 and CTE
We then tested whether the spatial arrangement of RTEm26 and CTE contributed to the synergistic effect. First, the reversal of the order of the elements from RTEm26-CTE to CTE-RTEm26 (Figure 5B) showed that the combination of the elements functions similarly in either configuration. Second, the 28 nt spacer between the elements was increased by insertion of a 325-nt spacer sequence (SP1), which led to a significant loss of synergy (Figure 5B). To exclude that the nature of the spacer RNA contributed to this effect, a different RNA fragment (SP2) was used (Figure 5B), resulting in a similar decrease in gag expression. Separation of the elements by shorter spacers of 202 and 100 nt led to gradual decrease in RTEm26-CTE activity (data not shown). Thus, the optimal synergistic effect requires the up-regulatory mutant RTE (RTEm26) and a functional CTE at close proximity.
The question arose whether multimers of CTE or RTE alone could achieve a similar effect. The presence of a CTE multimer has been reported to improve expression i.e. 4xCTE in a gag/pol reporter further elevated expression but this depended on the nature of the polyA signal [36], whereas multiple copies of the CTE had little or no effect in other mRNAs ([36], our own observations), suggesting that an effect of CTE multimers depended on the mRNA context. Using the gag reporter plasmid used herein, pNLgag, we found that two adjacent CTE elements also synergized reaching ~75% of the effect observed for RTEm26-CTE. In contrast, we found that RTEm26 does not synergize with itself. Thus, while the effect of CTE multimers is transcript dependent, the RTEm26-CTE mediated synergistic increase in gene expression was persistently observed using different mRNAs (Figures 1, 2, 3, 4). Most importantly, the use of RTEm26-CTE has another great advantage, because this combination avoids the presence of adjacent repeated sequences, which may cause plasmid instability during growth in bacteria.
The rather unexpected finding of this work was that the combination of two retroviral/retroelement-derived cis-acting RNA export elements, RTE and CTE, synergistically increased expression of different retroviral mRNAs that are otherwise poorly expressed (Figures 1, 2, 3, 4). Since the function of RTEm26-CTE is conserved in mammalian cells, their integration into expression vectors provides a potent tool to improve expression of poorly expressed, unstable retroviral mRNAs to levels otherwise only achieved via more cumbersome RNA optimization.
Whereas the main restriction retroviral mRNAs encounter is their nucleocytoplasmic transport, other mRNAs may have different restrictions. The question arises whether the RTEm26-CTE combination has any effect on the expression of genes or cDNAs, which have posttranscriptional restrictions other than those of the lentiviral mRNAs. No improvement of expression was found using either the RTE, the CTE or the RTEm26-CTE combination in a MuLV-derived retroviral vector [37], while insertion of the woodchuck element WPRE augmented expression of this MuLV mRNA. These data indicate that oncoretrovirus and lentivirus expression have distinct restrictions. We further tested whether the presence of these RNA export elements could counteract posttranscriptional control that is not exerted at the RNA transport level but only involves cytoplasmic control. We noted that these export elements, as expected, did not alleviate the downregulatory effect of the AU-rich element (ARE)-containing IL-3 mRNA using the GFP-IL-3 hybrid mRNAs as a model system [38]. Thus, this finding supports the specific mechanism of function of the RTE, CTE and RTEm26-CTE, namely nucleocytoplasmic export. For this reason, we tested RTEm26-CTE effect upon insertion into some of our already RNA-optimized HIV and SIV gag and env cDNAs vectors, whose mRNAs are efficiently exported leading to very high expression in cultured cells. As expected, we only found a less than 2-fold effect on this already optimized mRNAs. It remains to be tested whether export signals like the RTEm26-CTE could act as additional positive acting signals and mediate higher expression levels in primary cells, for example upon DNA vaccination of animals or using gene transfer vectors.
NXF1 provides a key molecular link between mRNA and components of the nuclear pore complex. A possible model to explain the synergistic effect of RTE and CTE is that the duplication of these export elements may provide an improved target for NXF1 resulting in more efficient nucleocytoplasmic mRNA transport. Using in vitro gel-shift assays, we found that the binding of NXF1 (aa 61–372) to radiolabeled CTE is competed similarly by both excess cold CTE as well as RTEm26-CTE RNAs (data not shown). These data indicate that NXF1 binds to CTE as well as to the RTEm26-CTE RNA targets with similar affinity. We have previously shown that NXF1 is not a high affinity binder of RTE when compared to the CTE [6], suggesting the role of a distinct cellular protein mediating RTE RNA export. It is plausible that this putative factor tethers the RTE-RNAs directly or indirectly to the NXF1 pathway. Therefore, it is likely that the putative RTE-binding protein and NXF1 may act cooperatively. Studies are on-going to delineate the detailed mechanism of function mediating this cooperativity.
mRNA expression is controlled at several steps including nuclear export, cytoplasmic trafficking and polysomal association. The use of strong mRNA export elements is a powerful tool to alleviate restrictions linked to nuclear export. For a subset of lentiviral mRNAs encoding gag, pol or env, posttranscriptional control has been shown at multiple steps of export and polysomal association. The presence of potent RNA export elements is sufficient to alleviate all of theses restrictions. Integration of RTEm26-CTE into lentiviral vectors will increase gene expression essential for applications such as in gene therapy that are otherwise only obtained through RNA optimization. For DNA-based vaccine vectors, it remains to be seen whether the presence of strong binding sites for the cellular mRNA transport machinery is of further advantage when introduced into primary tissues in animals as compared to cultured cells. In addition, these retroviral/retroelement derived RNA export elements provide unique tools to further dissect mechanisms involved in posttranscriptional regulation of viral and cellular genes.
The use of the combination of RNA export elements, comprising the up-regulatory mutant RTEm26 and the CTE, potently increase lentiviral gene expression.
The RTE, RTEm26, and CTE were inserted into pNLgag [23, 24, 39] between the gag gene and the 3'LTR and have been described [7]. RTE or RTEm26 was inserted into the SacII site located 5' to the CTE, generating pNLgagRTE-CTE and pNLgagRTEm26-CTE, respectively. In pNLgag RTEm26-SP1-CTE, a spacer sequence (SP1) of 325 nt from a synthetic HIV-1 tat gene (BamHI-XbaI from plasmid 32H) was inserted between RTEm26 and CTE. In pNLgag RTEm26-SP2-CTE, a spacer (SP2) from a different source (cat gene) of 325 nt was inserted. Similarly spacers or 202 and 100 nt were inserted. The bovine growth hormone polyadenylation signal was inserted between SalI and XhoI sites 3' to RTEm26-CTE replacing the 3'LTR. pNLcgag [24] is similar to pNLgag, except it lacks the major splice donor. pNL1.5E expresses the authentic HIV-1 env cDNA from the LTR promoter [40]. RTE, CTE and RTEm26-CTE were inserted as SmaI-XhoI fragment between the env gene and the 3' LTR into BlpI and XhoI digested pNL1.5E. The Rev-independent clones of NL4-3 [RRE(-)Rev(-), RRE(-)Rev(-)CTE, and RRE(-)Rev(-)RTE] have been published previously [6, 12, 41]. RTEm26-CTE was inserted into the XhoI site of the RRE(-)Rev(-) NL4-3. The SIVmac239 RRE(-)Rev(-)nefdelCTE is similar to the published SIVmac239 RRE(-)Rev(-)Nef(-)CTE [35] but contains an additional deletion of the remaining nef region 3' to the CTE [42]. RTEm26-CTE was inserted in the place of CTE. The GFP-IL-3 plasmid contains the IL3 3'UTR inserted 3' to the enhanced green fluorescent protein (GFP) gene in pFRED25 [43]. RTEm26, CTE, or RTEM26CTE were inserted between GFP and the 3'UTR. These elements were further inserted between the cDNAs and the polyadenylation signals of vectors expressing the RNA-optimized HIV-1 env (75 H).
Human HLtat, a HeLa-derivative producing HIV tat [44] or human 293 cells were transfected with 1 μg of the NLgag plasmids. HLtat provides Tat to activate gene expression from the viral LTR promoter. For transfection of 293 cells a tat expression plasmid, pBstat, was also co-transfected. We routinely analyzed 2–3 independent clones in duplicate determinations. Two to three days later, the cell extracts were analyzed for Gag expression using a commercial HIV-1 p24gag or the SIV p27gag antigen capture assay. Gag and Env production was also analyzed on Western immunoblot using plasma from HIV-1 infected persons, rabbit anti-HIV-1 env serum or SIVmac infected rhesus macaques [23]. Cotransfection of 0.8 μg of the GFP expression vector pFRED25 [43] served as internal control. Cotransfection of the secreted version of alkaline phosphatase SEAP [45] as internal control was used in some experiments and SEAP levels were determined from the culture supernatant using a commercial kit (Tropix, Inc.). Transfections of 293 cells were performed using FUGENE-6, whereas the Calcium-phosphate coprecipitation technique was used for HeLa cells. GFP-IL3 plasmids were transfected into NIH3T3 cells and analyzed by fluorescent activated cell sorting (FACS) as described [38]. Total and cytoplasmic polyadenylated mRNA was isolated and analyzed as described [12, 46]. Hybridization of the blots with a GFP probe was used to evaluate transfection and RNA extraction efficiency. Blots were quantitated using the STORM860 phosphoimager.
constitutive Transport Element
RNA Transport Element
Rev-Responsive Element
human immunodeficiency virus type 1
simian immunodeficiency virus
intracisternal A-particle retroelement
simian type D retroviruses
nuclear export factor 1.
We thank S. Lindtner for comments, our Werner H. Kirsten Student Intern program recipients C. Jodrie, A. Gainer, L. Kotani, T. Hudzik, and S. Sadtler for their contributions, L. Arthur and J. Lifson for antiserum, M. Lu for technical assistance, and T. Jones for editorial assistance. AvG and MR are contractors through SAIC. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.
Authors' Affiliations
- Grüter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi A, Wilm M, Felber BK, Izaurralde E: TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell. 1998, 1: 649-659. 10.1016/S1097-2765(00)80065-9.View ArticlePubMedGoogle Scholar
- Segref A, Sharma K, Doye V, Hellwig A, Huber J, Luhrmann R, Hurt E: Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. Embo J. 1997, 16 (11): 3256-3271. 10.1093/emboj/16.11.3256.PubMed CentralView ArticlePubMedGoogle Scholar
- Tan W, Zolotukhin AS, Bear J, Patenaude DJ, Felber BK: The mRNA export in C. elegans is mediated by Ce-NXF-1, an ortholog of human TAP and S cerevisiae Mex67p. RNA. 2000, 6: 1762-1772. 10.1017/S1355838200000832.PubMed CentralView ArticlePubMedGoogle Scholar
- Braun IC, Rohrbach E, Schmitt C, Izaurralde E: TAP binds to the constitutive transport element through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus. EMBO J. 1999, 18: 1953-1965. 10.1093/emboj/18.7.1953.PubMed CentralView ArticlePubMedGoogle Scholar
- Erkmann JA, Kutay U: Nuclear export of mRNA: from the site of transcription to the cytoplasm. Exp Cell Res. 2004, 296 (1): 12-20. 10.1016/j.yexcr.2004.03.015.View ArticlePubMedGoogle Scholar
- Nappi F, Schneider R, Zolotukhin AS, Smulevitch S, Michalowski D, Bear J, Felber BK, Pavlakis GN: Identification of a novel posttranscriptional regulatory element using a rev and RRE mutated HIV-1 DNA proviral clone as a molecular trap. J Virol. 2001, 75 (10): 4558-4569. 10.1128/JVI.75.10.4558-4569.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Smulevitch S, Michalowski D, Zolotukhin AS, Schneider R, Bear J, Roth P, Pavlakis GN, Felber BK: Structural and Functional Analysis of the RNA Transport Element (RTE), a Member of an Extensive Family Present in the Mouse Genome. J Virol. 2005, 79: 2356-2365. 10.1128/JVI.79.4.2356-2365.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Tabernero C, Zolotukhin AS, Valentin A, Pavlakis GN, Felber BK: The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J Virol. 1996, 70: 5998-6011.PubMed CentralPubMedGoogle Scholar
- Tabernero C, Zolotukhin AS, Bear J, Schneider R, Karsenty G, Felber BK: Identification of an RNA Sequence Within an Intracisternal-A Particle Element Able to Replace Rev-Mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1. J Virol. 1997, 71: 95-101.PubMed CentralPubMedGoogle Scholar
- Ernst RK, Bray M, Rekosh D, Hammarskjold ML: Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element. RNA. 1997, 3: 210-222.PubMed CentralPubMedGoogle Scholar
- Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, Hammarskjold ML: A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci USA. 1994, 91: 1256-1260.PubMed CentralView ArticlePubMedGoogle Scholar
- Zolotukhin AS, Valentin A, Pavlakis GN, Felber BK: Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes. J Virol. 1994, 68: 7944-7952.PubMed CentralPubMedGoogle Scholar
- Ernst RK, Bray M, Rekosh D, Hammarskjold ML: A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol Cell Biol. 1997, 17: 135-144.PubMed CentralView ArticlePubMedGoogle Scholar
- Izaurralde E, Spector DL: Nucleus and gene expression; The interplay of transcriptional and post-transcriptional mechanisms that regulate gene expression. Curr Opin Cell Biol. 2004, 16 (3): 219-222. 10.1016/j.ceb.2004.04.005.View ArticleGoogle Scholar
- Dreyfuss G, Kim VN, Kataoka N: Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol. 2002, 3 (3): 195-205. 10.1038/nrm760.View ArticlePubMedGoogle Scholar
- Vinciguerra P, Stutz F: mRNA export: an assembly line from genes to nuclear pores. Curr Opin Cell Biol. 2004, 16 (3): 285-292. 10.1016/j.ceb.2004.03.013.View ArticlePubMedGoogle Scholar
- Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR: The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989, 338: 254-257. 10.1038/338254a0.View ArticlePubMedGoogle Scholar
- Hammarskjöld ML, Heimer J, Hammarskjöld B, Sangwan I, Albert L, Rekosh D: Regulation of human immunodeficiency virus env expression by the rev gene product. J Virol. 1989, 63: 1959-1966.PubMed CentralPubMedGoogle Scholar
- Feinberg MB, Jarrett RF, Aldovini A, Gallo RC, Wong-Staal F: HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986, 46: 807-817. 10.1016/0092-8674(86)90062-0.View ArticlePubMedGoogle Scholar
- Schwartz S, Felber BK, Pavlakis GN: Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol. 1992, 66: 150-159.PubMed CentralPubMedGoogle Scholar
- Schneider R, Campbell M, Nasioulas G, Felber BK, Pavlakis GN: Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/Protease and particle formation. J Virol. 1997, 71: 4892-4903.PubMed CentralPubMedGoogle Scholar
- Cochrane AW, Jones KS, Beidas S, Dillon PJ, Skalka AM, Rosen CA: Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J Virol. 1991, 65 (10): 5305-5313.PubMed CentralPubMedGoogle Scholar
- Hadzopoulou-Cladaras M, Felber BK, Cladaras C, Athanassopoulos A, Tse A, Pavlakis GN: The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol. 1989, 63: 1265-1274.PubMed CentralPubMedGoogle Scholar
- Felber BK, Hadzopoulou-Cladaras M, Cladaras C, Copeland T, Pavlakis GN: rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci USA. 1989, 86: 1495-1499.PubMed CentralView ArticlePubMedGoogle Scholar
- Nasioulas G, Zolotukhin AS, Tabernero C, Solomin L, Cunningham CP, Pavlakis GN, Felber BK: Elements distinct from human immunodeficiency virus type 1 splice sites are responsible for the Rev dependence of env mRNA. J Virol. 1994, 68 (5): 2986-2993.PubMed CentralPubMedGoogle Scholar
- Maldarelli F, Martin MA, Strebel K: Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: Novel level of gene regulation. J Virol. 1991, 65: 5732-5743.PubMed CentralPubMedGoogle Scholar
- Schwartz S, Campbell M, Nasioulas G, Harrison J, Felber BK, Pavlakis GN: Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type-1 results in Rev-independent gag expression. J Virol. 1992, 66: 7176-7182.PubMed CentralPubMedGoogle Scholar
- Suh D, Seguin B, Atkinson S, Ozdamar B, Staffa A, Emili A, Mouland A, Cochrane A: Mapping of determinants required for the function of the HIV-1 env nuclear retention sequence. Virology. 2003, 310 (1): 85-99. 10.1016/S0042-6822(03)00073-4.View ArticlePubMedGoogle Scholar
- Lu X, Lewis N, Rekosh D, Hammarskjold ML: A 5' splice site is essential for rev and rex regulation of HIV envelope protein mRNA expression. Advances in Molecular Biology and Targeted Treatment for AIDS. Edited by: Kumar A. 1991, New York , PlenumGoogle Scholar
- Dangel AW, Hull S, Roberts TM, Boris-Lawrie K: Nuclear interactions are necessary for translational enhancement by spleen necrosis virus RU5. J Virol. 2002, 76 (7): 3292-3300. 10.1128/JVI.76.7.3292-3300.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Jin L, Guzik BW, Bor YC, Rekosh D, Hammarskjold ML: Tap and NXT promote translation of unspliced mRNA. Genes Dev. 2003, 17 (24): 3075-3086. 10.1101/gad.1155703.PubMed CentralView ArticlePubMedGoogle Scholar
- Hull S, Boris-Lawrie K: RU5 of Mason-Pfizer monkey virus 5' long terminal repeat enhances cytoplasmic expression of human immunodeficiency virus type 1 gag-pol and nonviral reporter RNA. J Virol. 2002, 76 (20): 10211-10218. 10.1128/JVI.76.20.10211-10218.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- D'Agostino DM, Felber BK, Harrison JE, Pavlakis GN: The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNA. Mol Cell Biol. 1992, 12: 1375-1386.PubMed CentralView ArticlePubMedGoogle Scholar
- Sodroski J, Goh WC, Rosen C, Dayton A, Terwilliger E, Haseltine W: A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986, 321: 412-417. 10.1038/321412a0.View ArticlePubMedGoogle Scholar
- von Gegerfelt AS, Felber BK: Replacement of posttranscriptional regulation in SIVmac239 generated a Rev-independent infectious virus able to propagate in rhesus peripheral blood mononuclear cells. Virology. 1997, 232: 291-299. 10.1006/viro.1997.8567.View ArticlePubMedGoogle Scholar
- Wodrich H, Bohne J, Gumz E, Welker R, Krausslich HG: A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression. J Virol. 2001, 75 (22): 10670-10682. 10.1128/JVI.75.22.10670-10682.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Hlavaty J, Schittmayer M, Stracke A, Jandl G, Knapp E, Felber BK, Salmons B, Gunzburg WH, Renner M: Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors. Virology. 2005, 341 (1): 1-11. 10.1016/j.virol.2005.06.037.View ArticlePubMedGoogle Scholar
- Benjamin D, Colombi M, Moroni C: A GFP-based assay for rapid screening of compounds affecting ARE-dependent mRNA turnover. Nucleic Acids Res. 2004, 32 (11): e89-10.1093/nar/gnh086.PubMed CentralView ArticlePubMedGoogle Scholar
- Solomin L, Felber BK, Pavlakis GN: Different sites of interaction for Rev, Tev, and Rex proteins within the Rev responsive element of human immunodeficiency virus type 1. J Virol. 1990, 64: 6010-6017.PubMed CentralPubMedGoogle Scholar
- Schwartz S, Felber BK, Fenyö EM, Pavlakis GN: Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J Virol. 1990, 64: 5448-5456.PubMed CentralPubMedGoogle Scholar
- Valentin A, Aldrovandi G, Zolotukhin AS, Cole SW, Zack JA, Pavlakis GN, Felber BK: Reduced viral load and lack of CD4 depletion in SCID-hu mice infected with Rev-independent clones of human immunodeficiency virus type 1. J Virol. 1997, 71: 9817-9822.PubMed CentralPubMedGoogle Scholar
- von Gegerfelt AS, Alicea C, Valentin A, Van Rompay KKA, Markham P, Marthas ML, Pavlakis GN, Ruprecht RM, Felber BK: Long lasting control and lack of pathogenicity of the attenuated Rev-independent SIV in rhesus macaques. submitted.
- Stauber RH, Horie K, Carney P, Hudson EA, Tarasova NI, Gaitanaris GA, Pavlakis GN: Development and applications of enhanced green fluorescent protein mutants. Biotechniques. 1998, 24 (3): 462-6, 468-71.PubMedGoogle Scholar
- Schwartz S, Felber BK, Benko DM, Fenyö EM, Pavlakis GN: Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol. 1990, 64: 2519-2529.PubMed CentralPubMedGoogle Scholar
- Berger J, Hauber J, Hauber R, Geiger R, Cullen BR: Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988, 66: 1-10. 10.1016/0378-1119(88)90219-3.View ArticlePubMedGoogle Scholar
- Zolotukhin AS, Tan W, Bear J, Smulevitch S, Felber BK: U2AF participates in the binding of TAP (NXF1) to mRNA. J Biol Chem. 2002, 277 (6): 3935-3942. 10.1074/jbc.M107598200.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
x Click Here to start receiving job updates in your mail box! Follow us on Instagram @jobzillanigeria
22 Jul 2016
Teaching Jobs in Abuja at Lordswill Academy, July 22nd 2016
by Jobzilla Nigeria at 7/22/2016 01:29:00 pm Tags: Abuja Jobs , Education and Training , Teaching Jobs
The Primary arm started off on 27th April, 2010 with Master Nic Brian Okoko as the pioneer pupil. Since then, enrollment has been on the increase with a total number of over two hundred and twenty (220) pupils/students presently.
Lordswill Academy consists of Pre-school (Foundation), Kindergarten, Primary, Junior and Senior Secondary schools.
We are recruiting to fill the positions below:
Job Title: Teachers
Mathematics, English Language, Igbo Language, Hausa Language, Guidance and Counseling, Trade, Home Economics, Music, ICT, Creative Art, Physical & Health Education, Basic Technology, Social Studies, Civic Education, Business Studies, Commerce, Government, Accounting, Agric Science, Biology, IRK, CRK, Literature-in- English, French, Primary and Pre-school Education, Chemistry, Physics, Economics.
Job Title: Head-Teacher
Job Title: Principal
Location: Abuja
Job Description
- We believe in the professionalism of our teachers and the positive impact they make on our scholars. We are proud of our long term success and measurable impact on our community.
- Currently we are in need of world class high performance teachers - focused on excellence and continuous improvement.
- We are passionate about education and all forms of learning. Teaching for us is the most fulfilling career.
- Interested candidates with suitable qualification and experience (Minimum of 2.1 in a relevant Bachelor's Degree, a PGCE [Post Graduate Certificate in Education] and a minimum of 3 years suitable teaching experience) are invited to apply.
- Passionate
- Creative
- Enthusiastic
- Flexible
- Collegial
- Everyday Learners
- Values Driven
- Broadminded
- Dedicated
How to Apply
Interested and qualified candidates should send their applications and resume/CV's to: email@example.com use the title of the subject/position you are applying for as your email subject.
Application should contain the following (photocopies):
- Birth certificate or age declaration
- Certificate of origin
- First school leaving certificate
- Computer literacy certificate
- Nysc discharge certificate
- Evidence of previous appointment & reason(s) for leaving
- Curriculum vitae
- Handwritten application
- Referees (previous employers)
- Two passport photograph
- Either of: National ID/International Passport/Voter's card
Follow @jobzillanigeria
You can also join our interactive and interesting JobZilla BBM Channel by clicking C00299C49.
Share this
Subscribe to: Post Comments ( Atom )


text:
Full of charming country style, the Dublin® River Plaid II Boot offers waterproof protection that's perfect for riders. Upper is crafted of waterproof Redskin leather with durable plaid nylon panels that feel like wool. A hidden top gusset and tie at the cuff allow for a comfortable fit adjustment over breeches or jeans. HBR® waterproof membrane to 160mm keeps feet dry. Rider Comfort System (RCS) insole provides all-day support and comfort with a padded heel cushion, cupped heel and arch support, sweat control lining and enhanced cushion layer. Lightweight and durable Tough Tech sole is designed to fit into a stirrup for riding. Adjustable lace at top for a perfect fit adjustment.
Dover Saddlery is proud to offer a 100% satisfaction guarantee on all of our products. We stand behind the quality of our products, and should you not be satisfied with your purchase, simply return it and we will refund, replace or exchange it for you.
100% Satisfaction Guaranteed - Your Money Back Any Time, for Any Reason.
Dover Saddlery understands your concerns regarding online security. We share in your concerns and therefore work hard with experts in the field to maintain a highly secure ordering environment. Along with our comment to security, we offer you a Secure Order Guarantee - this gives you 100% protection for your credit card transactions on Doversaddlery.com.
Our Secure Order Guarantee means that you will never have to pay any money related to unauthorized charges to your credit card account, as long as those charges are a direct result from a transaction on Doversaddlery.com.
How it works: If you experience credit card fraud, the Fair Credit Act specifies that your credit card company can hold you liable for no more than $50.00 in fraudulent charges to your account. Our Secure Order Guarantee will reimburse you for any amount up to $50.00 billed to you by your credit card company for unauthorized charges to your account, if those charges result from an order you placed with Doversaddlery.com. For this protection you must promptly notify your credit card company of fraudulent charges and follow their procedures as outlined in your credit card agreement.
Show us your equestrian style. #itsfromdover


text:
2003 Thunderbird has no power at all
I tried the lock this morning, will not work, key does not even click, no lights at all. Was okay 2
days ago
5 Answers
Was something possibly left on in the vehicle to drain the battery?
Nothing. I always turn the lights off and I don't use the radio alot since I am not familiar with it yet. Just got it 3 months ago.
I would check the battery terminals. Make sure they are tight and are clean of corrosion. If you have access to a charger, I would put a good charge on the battery and see if it takes a charge. You can also pull the battery and have it load tested at any auto parts store.
Yes, the first answer is "Right on"! In 33 years I have inadvertently left the interior dome light on 2 times in my '81 T-Bird. That's why I own a charger. Be careful when you push in/off the light switch that you don't rotate it.
I have a 2003 T-Bird. Love the car but it has turned out to be pretty high maintenance. If I don't drive it for 2-3 days, the battery drains and needs to be jumped. Suspecting a short in the system, I spent several thousand dollars sending my mechanic on a wild goose chase looking for it. He couldn't find anything, so I took it to the a Ford dealer service center. When I told the mechanic there about the problem, he said, "Oh, yeah — they do that." So don't let the car go more than a couple of days without starting it, and consider investing in some kind of trickle charger.
Your Answer
Looking for a Used Thunderbird in your area?
CarGurus has 1,051 nationwide Thunderbird listings starting at $4,500.
Search Ford Thunderbird Questions
Ford Thunderbird Experts
Larry Sturgis
Reputation 1,540
Tom Demyan
Reputation 1,490
Reputation 1,210


text:
Various regulatory changes within the credit reporting industry will soon begin to have an effect on your practice's receivables. Even if your practice does not report delinquent patient accounts, understanding them and ensuring compliance is key to maintaining recovery.
You just received a phone call from an upset patient because their overdue balance with your practice is affecting their credit, and they are trying to get a loan for new car. It is the mission of your healthcare organization to establish lasting relationships with your patients while providing the highest quality patient care, so you begin to wonder whether credit bureau reporting past due balances is really the right choice for your practice.


text:
Bill Gates quits Microsoft
Wow, end of an era. Bill Gates is stepping down from full-time Microsoft role to transition to full-time work at the Bill & Melinda Gates Foundation. Good for him, it's gotta be tough to leave the company you founded. But I can't believe this is because Microsoft needs executive changes – Gates wasn't the problem. Ballmer and Allchin are the problem. They booted Allchin last year, which was smart. But Ballmer's gotta be on his way out the door too. Ballmer is a sales guy who'd be just as happy back at Procter & Gamble selling detergent. He's the one that turned the company from a software company to a company that makes software. That's when Microsoft lost its edge.


text:
As a researcher, your job is to collect stories from real people to find actionable insights. However, instead you often find yourself spending more time on logistics than actually doing research. That sucks, right?
Dovetail helps you with the key steps of research: designing your study, collecting data, analyzing results, and sharing what you've learned.
Loved by over 200 organisations
Use Dovetail to learn about people's habits, culture, feelings, behaviours, and more.
Put yourself in your customer's shoes and see how they experience your product or service.
Learn more about your target market for a potential new product or service.
Check in regularly with employees and give them a way to provide suggestions and feedback.
Receive an SMS every weekday morning and reply with your stand-up message.
Send yourself email prompts to fill out your travel diary and simply reply with your entry!


text:
WARHOL, Andy
Place Published: Cologne
Publisher: Kerber
Date Published: 1997
Description: Introduction by Vincent Fremont. Essays by Robert Rosenblum, Kerber Verlag, Jablonka Galerie. Illustrated in both color and black & white. 77 pages. Tall, thin 4to, black cloth, d.w. Cologne: Kerber, (1997). A fine copy in a near fine dust wrapper.Jacket: Y
Binding: hardcover
Condition: fine
Language: English
Type: Books
Book ID: 255087


text:
Search nearby
Sponsored links
Routes nearby
Places of Interest nearby
Matched content
Hotels nearby
Restaurants nearby
Sponsored links
Location address: United Kingdom, Westminster
Number of texts: 1
St. James's Square is the only square in the exclusive St James's district of the City of Westminster. It has predominantly Georgian and neo-Georgian architecture and a private garden in the centre. For its first two hundred or so years it was one of the three or four most fashionable residential address in London. It is now home to the headquarters of a number of well-known businesses, including BP and Rio Tinto Group; to two London gentlemen's clubs, the East India Club and the Naval and Military Club; and to the London Library. Also based in the square is the world-famous think tank, the Royal Institute for International Affairs, better known as Chatham House. The square's main feature is an equestrian statue of William III erected in 1808.
Classify this POI
Link to route
Add this POI to your routes nearby