Involvement of the gene ZmHLB in transport hormones in the phloem
RBPs - RNA-binding proteins are involved in different aspect of post-transcriptional gene regulation. The Puf in S. cerevisiae are structurally related family of cytoplasmatic RBPs. The proteins got several consecutive repeats of the Pumilio homology domain (Pum-HD). Bind to the 3'UTR sequence. The five members bear 6-8 Puf (Pum-HD) repeats.
Your mission, should you choose to accept it, involves identify specific mRNAs that interact with the five members of the Puf family. Finding if there is a consensus sequence in the mRNAs that binds by those Puf members.
To summarise what we already know:
TAP-tag the Puf family
Based on the Rigaut et al. 1999 - built a 5 contracts with a sequence encoding two IgG-binding units of protein A, a speciﬁc protease recognition site, and a calmodulin-binding domain, was fused in-frame at the C-terminus of the respective open reading frame (ORF)in its original chromosomal location.
Isolate Puf Proteins
Will isolate Puf Proteins.
Extract cells, recover mRNA and labelling
Harvest the cells and extract the ribonucleoprotein complexes (IgG binding) RNA isolation from immunopurified samples. Labelling with fluorescent dyes
The mobility of GFP-tagged Puf1
Daphne Rosenfeld 03/28/2012
The fluorescence of a GFP-tagged protein is bleached in the region of interest.
Subcellular distribution of Puf proteins
Localization of the TAP-tagged Puf proteins with immunofluorescence with antibodies against the TAP tag.
- Add a link to existing resource
- Grant Proposal
- DRAFT - Assimilation of external DNA into mitochondrial DNA
- Lab presentations
- Lab Meeting Presentations 7-12-12
- Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease.
- Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.
- RNA Extraction
- Cell lines
- CRL-2245 - pgsC-605
20/04/2012 by Daphne
47.9K, No comments
22/04/2012 by Daphne
23.2K, 6 comments
23/04/2012 by Robert
1374K, 1 comment
- Uploaded files
2010 August Lab meeting
11/11/2010 by Daphne Rosenfeld
1.41 MB, No comments
2009 Grant Proposal
5/10/2010 by Daphne Rosenfeld
156K, No comments