Hi Victoria

There are a lot of possible explanations for this. You need to provide a few more details. First of all, did you clone each gene separately or did you clone them together (i.e. keeping the original operon structure)? Which bacteria are you using. How did you determine mRNA levels? Did you clone them into a vector or insert them into the genome?

Jesper

Hi Jesper,

I cloned the genes together, keeping the original operon structure. Cloned right from the start codon of one to the stop codon of the second one. I am working on Lactococcus and the genes were cloned from Sterptococcus. mRNA levels were determined by qPCR .

we have two clones here:1) In one the genes were integrated into the chromosome, where I obtained higher expression (mRNA ) levels of the second gene compared to the first.

2) Another work being carried where the genes were expressed in vector showed a different result. In this case , the 1st gene expression was 6-8 times higher than the second.

Thanks

Hi Victoria

(i) it can sometimes be difficult to compare expression data from plasmids with their chromosomal equivalents. Even low copy number plasmids are present in multiple copies and so, differences may be exaggerated or even masked.

(ii) the transcriptional landscape of bacteria seems to much more complex than previously believed. There is a paper from P. cossarts group about what they call the excludon (http://www.ncbi.nlm.nih.gov/pubmed/23268228) which are essentially overlapping transcripts all over the place.

(iii) there may be an alternative promoter within the operon which increases RNA levels of the second gene (test by northern blotting or bioinformatics + primer extension).

(iv) the mRNA may be subject to some kind of post transcriptional processing which could result in one of the genes being more stable than the other.

(v) my best guess is that it could be the qPCR setup. Two primer pairs for the same transcript may not give the same result in terms of Ct values. In our lab we have carried out qPCR on circular M13 RNA using several different primer pairs. In all cases there is a nice linear correlation between Ct values and the input of cDNA. However, not all pairs give the same Ct value for the same template concentration and so each of them has to be calibrated to their own standard curve. If you are normalizing your data to for example an endogenously expressed housekeeping gene, you cannot expect the same result for both genes. I recommend you try northern blotting using separate probes for each gene.

sorry for the long reply, hope you can use it.

Jesper

HI Jesper,

Thanks a lot. It is really helpful. I will check using northern blotting and also if there is a possibility of post transcriptional processing, I will go through the paper too.

Thanks a lot for helping

Victoria