Question

1. Describe in detail how a protein-encoding gene in a eukaryote is transcribed as mRNA, and what events happen to the mRNA before it can be translated into a protein.

2.Describe the principles behind and the applications of the following:

a) Northern blotting b) Site-directed mutagenesis c) DNase l footprinting d) Fusion protein vectors e) Sanger Sequencing of DNA

3.Describe six differences between DNA replication in bacteria compared with eukaryotes.

Answer 1

1. In eukaryotes, gene has a promoter to which the RNA polymerase binds with the help of various transcription factors to initiate transcription of the gene at a particular transcription site. the mrna produced is henceforth capped with 5 methyl guanosine and tailed with poly adenine tail. In addition the mrna is spliced to remove introns thw non-coding sequences between the coding sequences-exons

2) a) northern blotting is a procedure that identifies a particular segment of DNA. In this procedure the DNA is run on a gel which is then transferred on a nitrocellular membrane where it is hybridised with a probe. The probe has a complimentary sequence of the DNA of interest. The probe is also labelled (fluorescence tag or radio-tag). So when this probe finds its complimentary strand, it binds to it confirming the presence of a particular segment of DNA

b) site directed mutagenesis is a method to see how does a change or deletion or insertion in the particular sequence of the nucleotide

c) DNase I footprinting: in this experiment, the protein is bound to DNA and this protein DNA complex is subjected to digestion by DNase I. These digested fragments are then run on a gel along side the digested strand of DNA which was treated with protein (a control basically). the missing strands in the DNA-protein complex gel are the strands or the stretch where the ptoein basically binds.