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Fusion In Frame....?


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#1 indoubt

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Posted 14 September 2004 - 05:55 AM

hi guys!

i am new in this field, so i hope that "mature" people can show me the way. thanks in advance!

with fusion in frame does it mean that the nucleotide sequences of both DNA fragments i fuse with each other have to act as independent triplet unit? in this way we can maintain the polypeptide of both DNA fragments? in other words that their nucleotides don't come together to make the triplet codons from the start to the end no matter if the fusion is a C, N or randomly (please see example 1, below).

for instance if the nucleotides of DNA fragment 1 is k's and it is a plasmid and nucleotides of DNA fragment 2 is x's. i insert 2 into 1. should i before the fusion have to make sure that the DNA fragments i fuse have enough nucleotides each to make their own triplets codon?:

example 1: randomly fusion where i insert DNA fragment 2 in the middle of the plasmid:
....kkk kkk kkk xxx xxx xxx xxx kkk kkk kkk....
here we get inframe from the start to the end. is this the ideal fusion? we can see here that the nucleotide sequences of both fragments DON'T COME TOGETHER TO MAKE THE TRIPLET CODONS. they act as independent units. the polypeptides of both fragments are maintained.


example2: in this case i have used a fragment which does not have enough nucleotides to make the triplet codons for its own. that is why it come with the nucleotides of the plasmid to make the triplet codons. AND THIS IS A FUSION WE SHOULD AVOID, RIGHT? because the polypeptides of fragment 2 is changed, so do the plasmid.
...kkk kkk kkk xxx xxx xxk kkk kkk....


i try to shorten this down, but afraid that none will understand my point, so please be patient and read :)


thanks alot!

#2 jadefalcon

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Posted 14 September 2004 - 08:47 AM

ok, i have to admit that i don't understand you completely, but maybe i can be of some help anyway.

"fusion in frame" means the fusion of two proteins/petides. so you have to be careful with your cloning, so that the cloned product will have an ORF containing both protein sequences. Otherwise you will produce a frameshift, and that are most severe mutations, as you may remember.

Example:
(protein 1 = all glycine (GGA) , protein 2 = all methioneine (ATG) ):

GGA GGA GGA GGA ATG ATG ATG ATG
G...G...G...G...M...M...M...M
is fusion in frame

GGA GGA GGA GGA TGA TGA TGA TG
G...G...G...G...STOP
GGA GGA GGA GAT GAT GAT GAT G
G...G...G...D...D...D
is not in frame,


since the coding frame of the two genes is not the same, hence it will not translate into one fusion protein containing both proteins, but in a protein containing protein 1 and then some nonsense... (in the example Stops or Ds)

so you have to give great care for the correct sequence when doing this!

mike

Edited by jadefalcon, 15 September 2004 - 04:04 AM.

--- He who finds typos may keep them! ---

#3 indoubt

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Posted 14 September 2004 - 09:52 AM

ok, i have to admit that i don't understand you completely, but may i can be of some help anyway.

:D


hi mike! i was prepared that this was very messy :unsure: but i tried my best! thanks for your reply, i got the point.




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