Question

1. Dwarfism can be prevented in children that are unable to produce the human growth hormone (hGH) if the hormone is administered to them during development. Recombinant hGH for this use is produced by bacteria. Some of the steps involved in engineering bacteria to produce the hormone involved extracting RNA from people who did produce the hormone and then reverse-transcribing the RNA to DNA before cloning it into a bacterial plasmid. It would have been technically simpler to just clone the hGH gene from human DNA. Why were the RNA extraction and reverse transcriptase steps essential if the end goal was expression by a bacterial cell?

Answer 1

The gene for human growth hormone (hGH) is isolated from human pituitary gland.Insertion of whole hGH gene into plasmid vector and cloning in bacteria ( E.coli ) results into production of biologically inactive hormone because bacteria can translate the region of gene that are not translated in human thereby producing a prehormone containing an extra 26 aminoacids which might be difficult to remove.

So at first,segment of gene that codes for the first 24 aminoacids of hormone is constructed chemically from blocks of nucleotide. next mRNA for hGH is isolated from human pituitary gland tissue. Then using reverse transcriptase enzyme complimentary DNA (cDNA) is synthesized from mRNA. This full gene is cut with restriction endonuclease enzyme to remove first 24 gene.Then the  synthetic gene (gene for first 24 aminoacids) and cDNA are joined in order to obtain full gene with its own initiation codon (AUG). This full gene is ligated into a restriction site just downstream of Lac; promotor/operator region of the expression vector.​​​​​​