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Antibiotic resistance genes in a plasmid - How are they expressed? (Mar/26/2010 )

Dear all,

Here's an example I'd use to ask my questions regarding how genes are expressed in a plasmid. Now, I know that when we insert our genes into expression vectors (or any other vectors for that matter) the gene will be "read" and translated into its product provided it is in frame.

In the pBR322 below, I notice there are two antibiotic resistance markers going in opposite directions. Also, the origin is going in the counter clockwise direction. Say now I'm using tetracycline to select for my transformant, how'd the TetR gene be read since its on a collision course with the ori?

Or did I miss any lectures?

Thanks!

-dreamchaser_jc-

ori is for DNA replication only (oriGIN of replication)
if there is prokaryotic promoter before each antibiotic-resistance gene it does not matter what direction the whole promoter+gene lies

-vladooo-

dreamchaser_jc on Mar 27 2010, 12:44 AM said:

Dear all,

Here's an example I'd use to ask my questions regarding how genes are expressed in a plasmid. Now, I know that when we insert our genes into expression vectors (or any other vectors for that matter) the gene will be "read" and translated into its product provided it is in frame.

In the pBR322 below, I notice there are two antibiotic resistance markers going in opposite directions. Also, the origin is going in the counter clockwise direction. Say now I'm using tetracycline to select for my transformant, how'd the TetR gene be read since its on a collision course with the ori?

Or did I miss any lectures?

Thanks!




The origin is not transcribed by RNA polymerase, it is the starting region of replication for DNA polymerase. For any gene that is expressed, somewhere after the stop codon there will be a place where the RNA polymerase will fall off the DNA strand, which will either be dependent or independent of a protein called Rho (so you might hear about Rho-dependent or Rho-independent transcriptional termination). Typically what happens is there is a sequence downstream of the stop that causes the RNA polymerase to pause. A long enough pause will allow the polymerase to fall off. It may be hard to tell for sure where a Rho-dependent termination site is, but for Rho-independent, there will be a heavy AT-rich region. The A-T bond is the weaker of the base pairs (2 H bonds instead of 3 between G-C). The AT region causes some secondary structure, and knocks off RNA polymerase.

So, the transcription of tet and amp shouldn't be on a collision course, because their transcription should end relatively soon after their reading frames.

The above is for single genes. Operons are a little different, but the principle is the same. Rather than the RNA polymerase falling off after one gene, it keeps going (because of no Rho-dependent or independent termination), and transcribes other genes downstream. The beginning of these other genes is usually shortly after the end of the previous gene (or sometimes the start is before the stop of the previous gene). All the genes in an operon, however, will all be in the same direction, there would never be two "arrows" pointing in opposite directions in an operon.

It's been awhile since my bacterial genetics course, so some of the details above might be a little off, but I think the general idea is right.

-fishdoc-

Oh, so that's how things work. RNA and DNA.

Thanks vladooo and fishdoc. And fishdoc, I think it's GC rich instead of AT. Thanks again!

-dreamchaser_jc-