Chapter Summary

12.2

  • Transposons can be used to mutate genes and mark the defective gene with antibiotic resistance.
  • The location of a transposon insertion can be determined by sequencing from the end of the transposon across the insertion junction. The junction sequence is examined by online comparative BLAST analysis to identify the gene in the genome.
  • Annotations of the gene may provide clues as to the function of the protein.
  • Regulation of a gene can be determined by fusing that gene to an easily assayed reporter gene.
  • Operon fusions (also called transcriptional fusions) reflect transcriptional control of the subject gene.
  • Gene fusions (also called translational fusions) reflect transcriptional and translational control.

12.3

  • Northern blot technology uses labeled DNA from a specific gene to probe the levels and sizes of RNA made from that gene.
  • Southern blot technology uses labeled DNA from a specific gene to probe other genomes for the presence of that gene.
  • Western blot technology uses antibodies to specific proteins to detect the quantity and size of those proteins in cell extracts.
  • Electrophoretic mobility shift assay (EMSA) examines protein interactions with DNA.
  • Fusion proteins are made to easily purify proteins for biotechnology purposes.
  • Primer extension analysis uses labeled DNA probes and reverse transcriptase to identify the 5′ end of a transcript and thus the transcriptional start site of a gene.
  • DNase protection assays reveal the bases in a DNA sequence protected by a DNA-binding protein.
  • ChIP-on-chip technology can identify all of the sequences in a genome to which a given protein will bind.
  • Two-hybrid analysis uses hybrid proteins to detect protein-protein interactions.

12.4

  • Affinity-tagged proteins can expose interactions between many proteins in the cell, and can be used to build protein interaction maps.
  • Green fluorescent protein (GFP) fused to a cell protein allows tracking of that protein’s location or movement in a cell.
  • Multiplex PCR is used to detect multiple organisms in complex environmental or food samples.
  • Real-time PCR is used to quantify specific DNA or RNA molecules present in cell extracts.

12.5

  • Genes encoding insecticidal proteins from Bacillus thuringienesis have been engineered into plant genomes to protect crops from insect devastation.
  • Proteins from pathogenic bacteria and viruses cloned into fruits and vegetables may provide a costeffective means to vaccinate large populations.
  • Genetically engineered viruses are being tested as gene therapy vehicles to deliver DNA to correct inherited diseases in humans.
  • Phage display is a form of in vitro evolution in which DNA sequences encoding random peptides are cloned into phage capsid protein genes. Phage particles displaying peptides with a desired binding prop erty are pulled from the general phage population by biopanning.