Eukaryotic Gene Expression and PCR

Problems and Solutions in Cloning and Expressing Eukaryotic Genes

• We have learned how to clone a eukaryotic gene (human gene) into a prokaryotic organism (bacteria) but there are more hurdles in this process.
• As we learned in chapters 18 and 19, prokaryotes and eukaryotes control gene expression slightly differently:
  • Different promoters
  • Different control sequences
  • These are overcome by using a plasmid that has a prokaryotic promoter just upstream of the restriction site where the eukaryotic gene will be inserted.
    • This plasmid is called an expression vector.
    • The promoter, etc. will ensure that the eukaryotic gene is expressed.
• Another problem is that eukaryotic DNA has long noncoding regions (introns).
  • Since prokaryotes do not have to deal with introns they do not have the normal RNA splicing enzymes, etc. to remove these noncoding regions.
  • The solution has been to make artificial genes that lack introns.
  • Scientists isolate fully processed mRNA from eukaryotic cells (i.e., mRNA which has had the introns removed).
  • Reverse transcriptase (obtained from retroviruses) is added to make DNA transcripts from this mRNA.
    • This is called complementary DNA (cDNA) and is distinguished by the fact that it has genes with no introns
    • The cDNA can be added to a plasmid and then be expressed.
• Another problem is that eukaryotic proteins are heavily modified after translation.
  • Carbohydrate or lipid groups are added to the protein.
  • Prokaryotes cannot do this.
• Yeast cells are often used to express human genes.
  • Yeasts are eukaryotes (have most of the needed genetic machinery).
  • Single cell (easy to culture).
  • Divide rapidly (fast turnaround time).
  • One of the few eukaryotes that have plasmids!

Figure 16.13, Purves's Life: The Science of Biology, 7th Edition; Figure 20.5, page 369, Campbell's Biology, 5th Edition

Genomic Libraries

• The first figure below shows a “shotgun” approach to creating fragments of the genome—no single gene is targeted.
• Each clone in step 4 has a unique fragment of the genome.
• A complete set of clones, which together comprise the whole genome of an organism, is called a genomic library.
• These can be stored for later use.

Figure 20.3, page 367, Campbell's Biology, 5th Edition; unknown source

Amplifying DNA: the Polymerase Chain Reaction (PCR)

• A method for making many copies of a piece of DNA was discovered in 1983 by Bay Area researcher Kary Mullis.
• Starting materials:
  • Piece of DNA that has the sequence you want to amplify (targeted sequence).
  • Special kind of DNA polymerase that is heat resistant
    • Called taq polymerase because it was isolated from the bacteria Thermus aquaticus, which was discovered in hot springs in Yellowstone National Park!
  • DNA primers: short synthetic molecules of single-stranded DNA
    • They are complementary to the ends of the targeted DNA and they therefore determine what part of the DNA is to be amplified.
  • Four nucleotides: dATP, dCTP, dGTP, dTTP
    • The raw material for creating the new DNA
• An animation of the process is available.

Unlabeled figure, page 371, Campbell's Biology, 5th Edition