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5 questions without answers...!

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5 questions without answers...!

Postby djm129129 » Wed Aug 06, 2008 3:05 pm

Im throwing a major hail Mary here! If you can help to answer any or all of these questions I would be greatfully indebted. Your help here would prove invaluable. I have been working hard on this and at risk of losing two yrs of work. Thanks again for any help. Here we go:
1. A fusion gene for a membrane protein Y is fused to GFP. After transfection, where would I expect to see fluorescent signal first appearing? Is this based on a ribosomal location? Membrane?
2. An IRES was introduced between protein Y and the GFP sequence. What is the purpose of adding the IRES and is there a difference now with this new construct from the original GFP signal? I understand what an IRES is, but I cannot get around its use in this situation.
3. Can someone provide one difference between endothelial and monocyte cells in regards to the mechanism of receptor activation for VEGFR? I cannot find any trace of information about this. I know monocytes react to VEGFR-1.
4.How does the soluble form of VEGFR-1 in the extracellular spaces affect signal transduction? I am only aware of intracellular mechanisms.
5. If GFP is fused to the 3' end of a gene encoding a membrane protein with 5 membrane spanning domains, with 75 aa at the N-terminus lying on inside of plasma membrane, and 100 aa at C terminus on external surface of cell. I can draw the protein embedded in the membrane, but where will the GFP be located? I know GFP can be fused to both C and N terminals, but am I missing a basic point with the 3' information?
Thanks so much.
Respectfully,
D.
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Postby mith » Wed Aug 06, 2008 7:57 pm

1. They'll be produced at the ribosome, but that'll be unimportant compared to where they'll aggregate finally. I'm guessing your protein is addressed to the membrane?

2. Might be measuring activity vs location.

5. Transcription direction
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Re: 5 questions without answers...!

Postby snowcapk » Thu Aug 07, 2008 5:21 am

djm129129 wrote:1. A fusion gene for a membrane protein Y is fused to GFP. After transfection, where would I expect to see fluorescent signal first appearing? Is this based on a ribosomal location? Membrane?

Membrane of rough ER, assuming you do not disrupt the N-terminal ER signal sequence of the membrane protein while producing the fusion gene. Folded GFP won't be produced until the translating ribosome has associated with the rough ER, because SRP will block further elongation as soon as the N-terminal signal sequence is translated and until the SRP has associated with DP.
djm129129 wrote:2. An IRES was introduced between protein Y and the GFP sequence. What is the purpose of adding the IRES and is there a difference now with this new construct from the original GFP signal? I understand what an IRES is, but I cannot get around its use in this situation.

The purpose is to allow distinct ribosome populations to translate protein Y and GFP. The ribosomes that are translating protein Y will still be directed to the ER by SRP, but the ribosomes that translate GFP will not be ER-associated because there will be no signaling sequence at the N-terminus of the green fluorescent protein to bind SRP. GFP will be translated by free ribosomes and so fluorescence will be cytosolic, not ER/Golgi/PM-associated.
djm129129 wrote:4.How does the soluble form of VEGFR-1 in the extracellular spaces affect signal transduction? I am only aware of intracellular mechanisms.

VEGF that is bound to soluble VEGFR-1 (probably) cannot bind to cell surface VEGFR at the same time. The soluble form disrupts normal signaling through competitive inhibition of the ligand.
djm129129 wrote:5. If GFP is fused to the 3' end of a gene encoding a membrane protein with 5 membrane spanning domains, with 75 aa at the N-terminus lying on inside of plasma membrane, and 100 aa at C terminus on external surface of cell. I can draw the protein embedded in the membrane, but where will the GFP be located? I know GFP can be fused to both C and N terminals, but am I missing a basic point with the 3' information?

mRNA is translated 5'->3', so GFP will be fused to the C terminus and thus outside of the cell.
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