Recombinant DNA Technology

Recombinant DNA Technology

Promoter Reminder

• Promoter sequences are recognized by RNA polymerase holoenzyme
• Promoter lies adjacent to an activator-binding site
• RNA polymerase is composed of 5 polypeptides (α2ββσ) when it binds to promoters
• σ factor is the protein that recognizes specific promoter sequences
• Most important nucleotides are centered at –35 and –10 relative to the transcription start-site for promoters recognized by the classical "vegetative" E. coli sigma factor, σ70
• Promoter sequence: 5'- TTGACA—17 bp spacing—TATAAT-5 to 9 bp- START Codon

Genetic Control

• Genetic control of enzyme activity refers to controlling transcription of mRNA needed for an enzyme's synthesis
• In prokaryotic cells, this involves induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase
• Regulatory proteins are part of either an operon or a regulon
• Operon: a set of genes transcribed as a polycistronic message that is collectively controlled by a regulatory protein
• Regulon: a set of related genes controlled by the same regulatory protein but transcribed as monocistronic units
• Regulatory proteins may function either as repressors or activators

Genetic Control Groups

• Repressors: Regulatory proteins that block transcription of mRNA
• Activators: Regulatory proteins that promote transcription of mRNA
• Translational control (antisense RNA): A strand of RNA complementary to a mRNA coding for some polypeptide or protein

Lactose Operon

• E. coli will only synthesize the three enzymes it requires to utilize lactose if that sugar is present in the surrounding environment
• Lactose functions as an inducer
• In the absence of lactose, the repressor protein binds to the operator and RNA polymerase is unable to transcribe the genes for utilization of lactose
• When lactose is present, it binds to the allosteric repressor protein and causes it to change shape, allowing RNA polymerase to transcribe the genes required for lactose degradation

Gene Cloning

• Gene cloning involves four steps:
  • The vector DNA is cleaved with one or more restriction enzymes
  • The DNA to be cloned is joined to the vector by ligase enzyme, generating a recombinant molecule
  • The recombinant DNA is introduced into a host cell (e.g., bacteria)
  • The transformed colony is selected to amplify

Vectors

• Vectors: DNA molecules used to transfer foreign DNA fragments between cells
• Types of vectors:
  • Plasmid
  • Phage (virus)
  • Cosmid
  • Yeast Artificial Chromosome (YAC)
  • Bacterial Artificial Chromosome (BAC)

Creating and Screening a Library

• Isolation of genes encoding proteins is a fundamental objective of molecular biotechnology
• A DNA library for the selected organism can be generated by:
  • Using restriction enzyme to cut the DNA into smaller fragments
  • Inserting each fragment into a vector
  • Cloning and identifying the cells containing the target fragments
• The presence of the target DNA can be identified with a DNA probe

Cloning DNA Sequence That Encode Eukaryotes Protein

• Due to post-translational modification and the presence of introns, it is difficult to clone eukaryotic genes into prokaryotic ones
• To solve this problem, mRNA can be extracted and used as all introns regions are already removed
• mRNA can be isolated using a special isolation technique based on devising a chromatography method where the beads are attached to poly TTTTT tail, which will bind to the poly AAAAA tail present at the end of the mRNA
• Cloning DNA sequence that encodes eukaryotic protein involves:
  • Converting mRNA into dsDNA using reverse transcription and DNA synthesis
  • Cloning the cDNA
  • Identifying the transformed cell