5 replies to this topic

[Akila Wijerathna Yapa]

Dear All;
I just Wanna Help from you for a bit confusing problem that I have, I though you all are the best persons to get answer.

Here's an example I'd use to ask my questions regarding how genes are expressed in a plasmid.

1.) Why in different plasmids their some genes expressing in different directions? how it happens?

2.) In the pBR322 below, I’ve noticed that there are two antibiotic resistance markers going in opposite directions. Also, the origin is going in the counter clockwise direction. Could you please let me know now I'm using tetracycline to select for my transformant, how'd the TetR gene be read since it’s on a collision course with the ori?

3.) why in same cassette different directions are shown?

4.) In plasmids (e.g. TDNA of Agrobacterium) some of genes marked with <-----> , >------> symbols { same symbols show in transposons too ( <------> )}
Could you please tell me wht tells from these arrow heads?

I've attached some of related figures that I've seen and shown numbers according to questions.

[ascacioc]

1 - you have a promoter before your gene; the RNA polymerase recognizes the promoter and then it goes in the direction of the gene; different gene directions means that the gene is either on the upper or bottom strand; when the RNA polymerase binds to the promoter, it chooses which one is the coding strand of the two possibilities
2 - Both Tet and Amp genes have their own promoters (see above for direction) and terminators. Tet stops before the origin since there is a terminator before the origin starts; hence, there is no collision

Andreea

[Trof]

1) Why not? Everyones genes are in different directions, why should plasmids be exception? Some viruses even have the same sequence once read from one direction to make one protein and in the oposite direction to make a different one.

3) Not sure what are you refering to, but since it really doesn't matter in which direction a complete casette is (unless in special cases, when some ORF inside other gene can cause blockage of transcription) and usually plasmids were constructed by restriction and ligation, sometimes the ligation is not direction specific so two possible orientations can be created.

4) Arrows usually denote the orientation of repetitive sequences. That is important, sice the repeat direction can have different functions (tandem versus oposite).

[Akila Wijerathna Yapa]

ascacioc, on 14 November 2012 - 02:37 PM, said:

1 - you have a promoter before your gene; the RNA polymerase recognizes the promoter and then it goes in the direction of the gene; different gene directions means that the gene is either on the upper or bottom strand; when the RNA polymerase binds to the promoter, it chooses which one is the coding strand of the two possibilities
2 - Both Tet and Amp genes have their own promoters (see above for direction) and terminators. Tet stops before the origin since there is a terminator before the origin starts; hence, there is no collision

Andreea

Dear Andreea, Thank you very much for kind reply

[Akila Wijerathna Yapa]

Trof, on 14 November 2012 - 03:40 PM, said:

1) Why not? Everyones genes are in different directions, why should plasmids be exception? Some viruses even have the same sequence once read from one direction to make one protein and in the oposite direction to make a different one.

3) Not sure what are you refering to, but since it really doesn't matter in which direction a complete casette is (unless in special cases, when some ORF inside other gene can cause blockage of transcription) and usually plasmids were constructed by restriction and ligation, sometimes the ligation is not direction specific so two possible orientations can be created.

4) Arrows usually denote the orientation of repetitive sequences. That is important, sice the repeat direction can have different functions (tandem versus oposite).

Dear Trof; Thank you very much for your kind reply

[Akila Wijerathna Yapa]

Thank you very much for spending your valuable time on solving my confusing problems, Wish good luck for both of your career.

Akila