gene fusions and reading frames

Andre Hamel hamel at ccu.umanitoba.ca
Tue Nov 30 11:26:37 EST 1993



I recommend making use of the guidelines determined by Marilyn Kozak
(for example, "Structural Features in Eukaryotic mRNAs That Modulate the
Initiation of Translation" Journal of Biological Chemistry, Vol.266 No.30,
October 25, pp 19867-19870, 1991). She's done excellent systematic emperical
characterizations of translation start sequences, and concluded that the
following (now deemed by many/most as "Kozak's Rules") is optimal for
translation in vivo:

GCC(A/G)CCAUGG
          ^^^

There are also upstream NON-AUG sequences in eukaryotic mRNAs that initiate
translation, in addition to initiation at first AUG codon.

You could also do a literature search for more recent work in this field. 

best wishes,
                            ********************
Andre Hamel                              email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services          lab tel.: (204) 945-7630
545 University Crescent,                   FAX:(204) 945-8062 
Winnipeg, Manitoba, 
CANADA   R3T 5S6            ********************


>We have a question: when making a promoter-gene fusion (for example 35S - CAT),
>which logic has to be followed to be sure that our gene's  ATG  will be the act
>ual translational start? someone told us that the only thing that matters is th
>at our ATG must be the first encountered after the transcriptional start...
>The problem is that, in vivo, evidences exist showing that this is not true.
>For example, take this:
>Bowman-Birk prot. inhib. CTTTTCC-TTGTGGGGGTTACTAATGCACACATGGAA.....
>        VS                                      ---      no
>C-II prot. inhib.        CTTTTCCTTTGTGGGGGTTACTAATGCACACATGGAA.....
>                                                no      ---
>In this case, a base insertion upstream the first ATG shifts the translational
> start to the second ATG. Moreover, many bases upstream these codons, some o
>ther ATGs are present on the mRNA sequences. Now the questions are:
>- does the reading frame start from the 1st coding ATG or from somewhere upstre
>am, in the transcribed sequence (e.g. the +1)?
>- Is there any general rule to follow in order to decipher this reading frame,
>or the only thing to do is to see what happens in vivo?
> 
>We sincerely apologize if the questions may sound stupid or useless or ignorant
>but nobody 'round here (Ph.D.s and professors included) has been able to give u
>s a clear answer. Thanks a million|
> 
>Massimo Delledonne & Lorenzo Frigerio
>Istituto di Genetica Vegetale
>UCSC Piacenza
>delle at ipcucsc.bitnet
>botanica at imicilea.cilea.it
> :-) :-) :-) :-) :-) :-)



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