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Sequence Translation is used to translate nucleic acid sequence to corresponding peptide. Protein Sequence Back-translation Backtranseq (EMBOSS) EMBOSS Backtranseq back-translates protein sequences to nucleotide. Starting the protein synthesis machine: eukaryotic translation initiation. Regulation of m. RNA translation during mitosis. There were two major concerns with the manuscript: 1) whether the Cdk. G2 phase might influence the conclusions of the study, given the known Cdk. EBP1 and e. IF4. G, and 2) why our data seems to be at variance with prior finding in the literature. We regret that our prior manuscript was unclear on these two issues, but we feel that there are strong reasons to mitigate both concerns. A detailed discussion can be found in the response to the referees. We feel that we have rigorously addressed the comments from the review process. We also feel that this study provides the first “big picture” view of translational regulation during mitosis and thus will be a well- regarded paper in e. Life. Reviewer #1: 1) Whereas a FACs analysis of the G2 sample is provided in. Figure 1. B, what do representative analyses of the M and G1 samples look like? We have included new FACS analysis of sample purity for both M and G1 cells in Figure 1. C, which confirm that these samples too are very pure. Emi. 1 (aka FBXO5) (Gene ID: 2. UTRs. Which one(s) is/are expressed in RPE- 1 cells? Which one was used to generate the reporter constructs? Which one is present during M phase? Do they all impart translational control? In fact, there seem to be 3 different proteins made via alternative exon 1 usage. The effects of differential isoform expression are interesting, and we have performed additional experiments to address this question. We have mapped the region in the UTRs of Emi. UTR of Emi. 1. Since the 3’UTR is identical in all Emi. We have now included this new data showing the involvement of the 3’UTR of Emi. Figure 3–figure supplement 1. D. For clarity, we have also included the Emi. UTR sequences (as well as the sequences of all other UTRs used in this study) in the Supplemental Methods section. The Emi. 1 3’UTR sequence that we used for our fluorescence translation reporter is identical to that of all known Emi. The 5’UTR used in our reporter assay (Figure 3. D) covers the complete 5’UTR of Emi. RNA- seq data relatively well. Visual representation of these results are now included in Figure 3–figure supplement 1. C. We also analysed isoform expression in G2 versus M, and found that the same Emi. Figure 3–figure supplement 1. C). Independent validation of the change in translation of the native transcript would significantly strengthen the author's conclusions here (and in. Figure 4. D)We note that, in fact, we do have two independent lines of evidence that Emi. First, ribosome profiling revealed that the endogenous m. RNA is translationally repressed. Second, we confirm these results using the fluorescence reporter and genetic elements of the Emi. RNA. 3) The Fluorescence- based translation reporter is interesting. Search for articles by this author Affiliations. Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Molecular Biology and Genetics, Howard Hughes. LincRNA-p21 suppresses target mRNA translation. Yoon JH(1), Abdelmohsen K, Srikantan S. National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore. Protein Biosynthesis* Proteolysis. AMPK is a trimeric protein with the . VA merit review program, American Diabetes Association, Juvenile Diabetes. Does trimethoprim affect m. RNA levels? Long term treatment of cells with trimethoprim does not affect m. Cherry levels, which indicates that the m. RNA levels are not affected by trimethoprim treatment. This is confirmed by a previous study showing that trimethoprim has no detectable effects on m. RNA levels (Han et al., 2. Nat. Methods). What is the efficiency of the P2. A “Stop N Go” sequence (Figure 3. A)? For m. Cherry to be completely independent of the degron, it has to be close to 1. Does one see GFP- m. Cherry fusions by Western blot? The P2. A sequence indeed confers separation of both protein with very high efficiency, generally around 9. Kim JH et al., 2. Plos One). This is confirmed by the fact that m. Cherry levels are not detectably affected by TMP addition in our experiments (See Figure 3. B). A note has been added to the manuscript that refers to the high efficiency of the P2. Custom mRNA Synthesis. Efficient translation of the mRNA into protein also requires a poly(A). After translation the protein will usually. Read about the Educational Outreach Program. MRPP3 mRNA and protein levels are reduced. A sequence. 4) Exactly which 5' UTRs were tested in. Figure 4. D? If more than one 5' (or 3'UTR) is known for each specific transcript, then assaying only one out of a possible set of combinations needs to be justified (see my comment #2 above). For ARHGAP5 (Gene ID 3. Figure 4. D? Which one is relevant for RPE- 1 cells? Throughout this study we use the full length 5’ and 3’ UTRs; the sequence of each is now provided in the Supplemental Methods section. Furthermore, we have now included a new figure (Figure 3–figure supplement 1. C), which shows the RNA- seq data across the Emi. UTRs. Importantly, we have also included new data showing that the 3’UTR of Emi. Figure 3–figure supplement 1. D). Since the 3’UTR is identical in all Emi. Emi. 1 3’UTR in our RNA- seq dataset (Figure 3–figure supplement 1. C), our new results show that all Emi. We have also included the sequence of the ARHGAP5 UTRs used in the experiments described in Figure 4. D- F. It is important to note that the goal of these experiments was to show that translational repression, rather than another function of Emi. UTR (like m. RNA localization), was responsible for increased APC activation. The ARHGAP5 UTRs are therefore solely used as a tool to suppress Emi. Figure 3. D), rather than to study ARHGAP5 translational regulation per se. In these experiments it is therefore not critical to know which ARHGAP5 isoforms are endogenously expressed in RPE1 cells. We have adapted the manuscript to indicate this more clearly. Finally, as a general comment, we do acknowledge that it is difficult to exclude that specific isoforms may lack the genetic element that confers translational regulation. However, it is important to note that the level of translational regulation in our ribosome profiling is based on the sum of the reads over all m. RNA isoforms. Thus, if the m. RNA of a particular gene is translationally repressed on average by 5- fold in our dataset, it is possible that one isoform is repressed while another isoform is not regulated. However, this unregulated isoform cannot exceed 2. RNA for that gene, and therefore makes only a minor contribution to the overall synthesis of the encoded protein. UTR is fused to m. Cherry- Emi. 1 to render it under M phase control, but there are 3 different transcription start sites for this gene, which one was used? Are the others also present in M phase? Do they impart translational control? See our response to questions 2 and 4. What is the efficiency of the P2. A “Stop N Go” sequence (Figure 3. A)? For m. Cherry to be completely independent of the degron, it has to be close to 1. If Westerns are performed, does one see GFP- m. Cherry fusions? See our response to question 3. Does stabilization of m. Cherry- Emi. 1 produced from Emi. ARHGAP5 5'UTR prevent or delay the decrease in GFP- Aurora A seen at 1. We have included a representative movie following GFP- Aurora A for several hours after mitosis, which reveal that m. Cherry- Emi. 1 under regulation of control UTRs prevents, rather than delays GFP- Aurora A degradation (Movie 1). Aurora A is normally degraded (within 1. Emi. 1 is under control of either Emi. ARHGAP5 UTRs. Reviewer #2. Mol Cell Biol, 2. CDK1 phosphorylation of these two translation initiation components dramatically affects their translational activity. Therefore, the criticism against microtubule targeting drugs may also be true for CDK1 inhibitors. How can the authors be sure that the data obtained are not due to side effects on translation initiation instead of cell cycle- dependent events? This important comment applies to all the comparisons made with G2- arrested cells as they have been obtained by incubating cells with the CDK1 inhibitor for 1. Unfortunately, the main message of the paper arises from comparisons between mitotic and G2- arrested cells. We agree with the reviewer that this is a critical point for interpreting our study. However, we strongly believe that the use of this inhibitor does not impact the major conclusions of this manuscript, as described below. We have re- written the manuscript to clearly explain the implications of using this drug to synchronize cells. CDK1 is indeed a critical kinase of several general translation factors, as described by the reviewer. However, throughout most of the G2 phase, CDK1 is completely inactive (Jackman et al., Nat. Biol., 2. 00. 3; Gavet & Pines, Dev Cell, 2. CDK1 occurs immediately prior (. Thus, our G2 sample, which is created by addition of a highly specific CDK1 inhibitor (Vassilev, PNAS, 2. G2 state very closely with respect to low CDK1 activity. Importantly, the notion that CDK1 is not active in phosphorylating general translation factors in G2, is fully supported by experimental data showing no significant phosphorylation of 4. E- BP1 in G2 phase (Heesom et al., 2. Curr. Biol). When we release cells from G2 by removing the inhibitor with our synchronization protocol, then cells procede into M with high CDK1 activity (mitotic entry has an absolute dependence on CDK1 activation, so the fact that the cells enter mitosis in our synchronization protocol proves that CDK1 becomes fully activated (Lindqvist et al., JCB, 2. Phosphorylation of 4. E- BP1 and e. IF4. GI by CDK1 then occurs during mitosis (Shuda et al., PNAS, 2. Dobrikov et al., MBo. C 2. 01. 4) at a time when CDK1 activity is very high. These phosphorylation events could contribute to the effects on translation that we observed; however, these effects occur both in unperturbed M cells and the synchronized M cells in our study, which both have robust CDK1 activity. In summary, the use of a CDK1 inhibitor to arrest cells in G2 followed by its washout closely mimics the CDK1 levels in unperturbed cells in their G2, M and G1 states. We apologize for the confusion due to the minimal description of our synchronization method and its implications in the initial version of the manuscript. We have now included detailed explanation of this critical point in this revised manuscript (in the Results and Discussion sections) and we have cited all the papers mentioned above. A second important point is that the conclusions we draw based on our comparison between G2 and M cells are fully supported by our comparison of M and G1 cells, which both have no drug present. DJ & Dance Music, Tracks & Mixes. Scooter - Jumping All Over The World (Official Video HD) Kontor.TV. Subscribe Subscribed Unsubscribe 3,833,160 3M. Scooter - Best Hits (90's. Darmowa wyszukiwarka plik. 2 - 100 Greatest Hits. Scooter - Greatest Hits (2010). Zippyshare.com is completely free, reliable and popular way to store files online. We offer fast download speeds. The maximum filesize for a single file is 200 MB. 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Intuit Releases Quicken Mac 2. OS X Lion Compatible. FWIW, I only know about this actually happening thanks to Glenn Fleishman's tweet a few minutes ago on Twitter. Maybe tomorrow others will catch on? Any indication how Mountain Lion might work with the Quicken for Lion 'patch'? OS X El Capitan features new options for managing windows, smarter Spotlight search, app enhancements, and faster performance. Open Menu Close Menu; Apple; Shopping Bag. Apple Support Communities / Mac OS & System Software / OS X Mountain Lion. Apple released OS X Mountain Lion. Home > Blogs > VMware Fusion Blog. OS X Mountain Lion Released to the Masses! Or is Intuit deliberately obscure? I can't imagine how I'm going to break this news to her.. She reports that it works great. Can't be for the $1. I held off upgrading to Lion solely because of the Quicken problem. I tried Quicken Windows, using Parallels on my Mac, and I tried i. Bank. I had so many problems with file conversion that I was ready to just chuck all of my years of Quicken data and start all over; then the Lion fix was announced, so I waited for it. Today’s patch now allows for the software to run under Lion. There’s also a download button titled “Quicken Mac Lion 2007 DOWNLOAD for Windows”. OS X Mountain Lion; Mac OS X 10.5.6; OS X Lion; OS X Lion; Mac OS X 10.6.7; Shift It; OS X 10.8.1 Update; Mac OS X 10.6.4; Java Runtime Environment; More Advertisement. If you've been waiting to make the jump to OS X Lion or Mountain Lion. Quicken 2007 and Lion will play nice for. X Lion was released in 2011: Quicken. Intuit Releases Lion-Compatible Quicken 2007 for Mac. Lion-compatible Quicken 2007 for Mac is available either by direct download or on CD. I still have to wait while it considers some entries, but it's no longer painfully slow. Now that the Mint people are in charge of Quicken I'm cautiously optimistic that we'll be seeing more Mac love from Intuit. But this Intuit situation (amongst a few other things) has kept me from upgrading my Mac Pro, which apart from being my . I've held off upgrading to Lion because I have way too much data in Quicken to abandon. I'm sure it's too much to import into another program and, besides, no other program handles paying bills from within the software. That's a requirement for me. I'll wait a week or two to make sure there aren't too many horror stories and then I'll upgrade my Quicken. No e- mail from Intuit despite receiving one from them last December. The Intuit store will only allow those with US and Canadian addresses to buy it so, as a Brit, that counts me out. Will it run on OS 1. The Intuit page doesn't say. What is Intuit's policy on the future of Quicken for the Mac. If there is none then we all might as well abandon it now. Looks like I'll have to stay where I am until there's a modern version for the Mac (#naive. Hope). Anyway, I hope that my purchasing the stopgap version will encourage Intuit to develop a modern version. The first answer showed the tech support people didn't know anything about Mountain Lion. After lots of explaining, I then got this reply. Once Apple launches this OS, our product engineers will test the program with the same Mountain Lion OS and we will inform to all our customers about the same. That program has QIF export, and I'd thought that might allow me to switch back. Can Lion- compatible Quicken import QIF data? I am currently using Quicken 2. I placed a used i. Mac that I had retired on my desk next to my Mac Pro which was upgraded and ran the i. Mac on Snow Leopard solely because of Quicken. After 2. 0 years, I really didn't want to shift to another financial app. I downloaded the new Quicken 2. Lion and can run it on my Mac Pro and am happy again. However, I will save the Snow Leopard i. Mac until I find out what happens long range. Why didn't they do this earlier and preserve good will? Time to look at Money. Works. It's cross platform and works for several countries. Intuit is working to solve the problem but no fix date has been stated. If Intuit had brought out a fresh Quicken 2. Lion, I would have considered using it. This reeks of a desperation move for Intuit. Intuit knows this and ask for your Console crash file. No word on when they will fix! No, not quite as polished as Quicken was 1. Get i. Bank most of all to send Intuit a message. They have been nothing short of hostile to Mac users for 2. Why would you stick with an abuser? I'm looking for something to switch to, and I'll switch even if they come out with a Lion- compatible 2. Mac - - which, apparently, they won't. I have waited to upgrade to Lion.. After ignoring Mac users for almost a decade you want to charge me $1. I switched to i. Bank and am happy. They appear to care about us. The new version runs on Snow Leopard or later. This version of Quicken 2. Power. PC emulator is even older than that would have been. Surely selling Quicken 2. Windows bundled with Cider would be a simpler strategy with more of a future in terms of likely updates. It looks like it was all recompiled as Intel. Now, why Intuit previously said that recompiling as Intel was not feasible, and how they ended up overcoming whatever obstacle there was, is a valid question. I put the old Quicken 2. Lion compatible Quicken 2. Back to i. Bank. Installed new Quicken for Mac 2. Lion release. Download of stock quoted are entered into previous day and current day quotes are registered as no change. But then I'm a naive customer who expects marketing blurb not to lie : ). I've been running the patched version for a couple of weeks now (was able to open my data file with info back to 1. I noticed was that downloading mutual fund quotes to my portfolio was off by one day. 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One of the nice features of C++ is that you can give special meanings to operators, when they are used with user. You can hopefully see that this would wreak havoc on your program. Overloads and templates; Overloads and templates Overloaded functions In C++, two different functions can have the same name if their parameters are different. Program structure: Statements and flow control; Functions. C++ Programming Language. Program Notes: We overload the +. Write a C++ program to Operator Overloading Using a Friend Function. Write A C++ Program For Addition & Multiplication Of Two Matrices By Overloading. Write a C++ program to Operator Overloading Using a Friend Function. B.6 — New virtual function controls: override, final, default. 56 comments to B.6 — New virtual function controls: override, final, default, and. I don’t program in Python so I’m not aware of what tutorials. An override method provides a new implementation of a member that is inherited from a base class. 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