Question

One can take a fragment of genomic DNA carrying a eukaryotic gene and clone it into a bacterial plasmid, containing an origin of replication and a selectable marker gene. Although the eukaryotic DNA can be stably replicated in the bacteria, the gene is often not expressed. Explain four possible reasons why this is the case.

Answer 1

1) May be gene is not correctly oriented in the plasmid due to ligation in reverse direction. So the gene being read in reverse which is a totaly different sequence.

2) Bacteria lacks nucleus where transcription happens in eukaryotic cells. There the eukaryotic mRNA will undergo post transcriptional modifications too, inorder to protect the mRNA from cellular nucleases. So in the absence of all these requirements the expressed mRNA may be prone to degradation in bacteria.

3) Eukaryotic genes contain noncoding regions called introns which are removed by splicing in eukaryotic cells. Introns are absent in prokaryotic genes and there is no splicing mechanism in bacteria. So presence of non coding region may be hindering the translation process of the mRNA to protein.

4) The eukaryotic proteins undergo post translational modifications like glycosylation, phosphorylation etc. This mechanism is absent in bacteria. So in the absence of this modification the protein may be unstable in bacteria and is being degraded.