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Find video protocols related to scientific articles indexed in Pubmed.
Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.
PLoS ONE
PUBLISHED: 04-26-2010
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We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to ?-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3dAMP), but sensitive to cordycepin 5-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3 end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).
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Multiple sequences and factors are involved in stability/degradation of Awnt-1, Awnt-5A and Awnt-5B mRNAs during axolotl development.
Dev. Growth Differ.
PUBLISHED: 02-16-2010
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Following fertilization in amphibian, early cleavage stages are maternally controlled at a post-transcriptional level before initiation of zygotic transcriptions at the mid blastula transition (MBT). We document the expression levels of the axolotl Awnt-1, Awnt-5A and Awnt-5B genes as well as the adenylation states of their corresponding mRNAs from the end of oogenesis until the tailbud stages. Awnt-1/-5A RNAs are stable until MBT then degraded before gastrulation. Awnt-5B RNAs are degraded at fertilization and zygotically expressed after MBT with high level expression from gastrulation. Estimation of the poly(A) tail lengths reveals no direct link between deadenylation and degradation periods for each Awnt transcript. To investigate the molecular mechanisms involved in Awnt-1/-5A/-5B RNAs stability, synthetic full-length or 3 untranslated region (UTR) Awnt RNAs progressively deleted from their 3 end were microinjected in axolotl oocytes, unfertilized and fertilized eggs. We identified degrading and stabilizing sequences in the 3UTR whose activities depend on the cellular context and are also modulated by the 5UTR and coding sequence within each RNA. Using axolotl nuclear extracts from stage VI oocytes, we further produced evidence of destabilizing factors targeting the Awnt-5B RNAs. Altogether, these results show that oocyte maturation and late cleavages following MBT are two important periods when axolotl Wnt RNAs are highly regulated.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.