Question

1. Molecular cloning has made it possible to take a DNA fragment from nuclear DNA, containing an entire gene, and insert it into a cloning plasmid just behind a phage promoter. This means that the cloned gene can be transcribed in a test tube using a commercially available phage RNA polymerase protein.

1. If you were to use agarose gel electrophoresis to separate RNA according to size, would the RNA produced in the test tube show any differences with RNA from the same gene produced in leaf tissue? Explain

b) Describe the molecular steps that are responsible for this difference.

c) What kind of differences would you be able to detect that is not possible to detect by agarose gel electrophoresis?

Answer 1

1) TNA produced in test tube differs from the RNA synthesized in leaf ( in vivo).

In vivo system - process post transcriptional modifications take place ( splicing) , which involve removal of intron ( non coding region) and ligation of exon ( coding region) . This mature RNA has something less length than premature RNA. In test tube , pre mature protein will not undergo splicing process thus have large size than in leaf synthesized protein.

B) splicing process invove rmexcidion of intron and ligation of exon.

Protein involve in thus process are known as SNRPs.

1) SNRPs bind with introns, and from spliceosome and introns loop out.

2) introns are excised , and exon are ligated

3) resulted RNA is mature and will translated to form protein.

Exon- T Intoon Exaniz pre-mRNA Splicatore SNKPS Intron - Mature Rast

C) after synthesis of protein , some pist translational modifications occur in protein. These are phosphorylation, disulphide bond formation etc. In these changes length of the protein will remain same , only modifications occur in amino acids present in the protein. So these kind of modifications or differences can not be possible to detect with agarose gel electrophoresis.