Coupling the expression of a gene with an easily assayable reporter gene provides a simple genetic trick for studying the regulation of gene expression. Two types of fusions between a gene and a reporter gene are possible: operon fusions place the transcription of a reporter gene under the control of the promoter of a target gene but the translation of the reporter gene and target gene are independent; gene fusions place the transcription and translation of a reporter gene under the control of a target gene, and result in a hybrid protein. Such fusions can be constructed in vitro using recombinant DNA techniques or in vivo using transposon derivatives. Many different transposon derivatives are available for constructing operon and gene fusions, but two of my favorites are (1) Mu derivatives that form operon and gene fusions to the lacZ gene, and (2) a Tn5 derivative that forms gene fusions to the phoA gene.
Mu is a transposon that can insert at essentially random sites in the E. coli or Salmonella chromosome. When Mu inserts into a gene it disrupts the gene and hence causes a mutation. Mu insertions are also polar on downstream genes in an operon.
Derivatives of Mu have been constructed that carry the lac operon (without its promoter) near one end of Mu (Casadaban and Cohen, 1979; Castilho et al., 1984). When these Mu derivitives (Mud) are inserted in a gene in the correct orientation, the lac genes are expressed from the promoter of the mutated gene. Hence, expression of the lac operon is directly proportional to expression of the mutated gene. Since expression of the lacZ gene can be easily detected on indicator plates and quantitated by assaying B-galactosidase activity, the expression of the mutant gene can be easily studied in vivo.
Two types of Mud fusion vectors are available:
Mud insertions. Mud can insert into a gene in either a "forward" or "reverse" orientation as shown in the figure below.
If Mud is inserted in the "forward" orientation (i.e. the lac operon faces the same direction the gene is transcribed), a Mud operon fusion is formed and the lac operon is expressed. (D) If it is inserted in the opposite, "reverse" orientation, transcription from the gene runs into the wrong end of the Mud so the lac operon is not expressed.
Mud fusions that express the lacZ gene can be identified on plates containing the indicator 5-bromo-4-chloro-3-indoyl-B-D-galactoside -- usually abbreviated as X-gal (or sometimes Xgal) for simplicity. X-gal is a colorless lactose analog that is a very sensitive indicator of B-galactosidase activity. When X-gal is cleaved by B-galactosidase a insoluble deep blue colored dye is produced.
Alkaline phosphatase is encoded by the phoA gene in E. coli. The wild-type phoA gene has a signal-sequence allowing export of alkaline phosphatase into the periplasm where it is active. Due to the highly reducing environment, alkaline phosphatase is not active in the cytoplasm. Alkaline phosphatase activity can be detected on solid media containing X-P (a colorless compound cleaved to form a blue colored compound like Xgal).
Gene fusions to phoA provide an assay for extracytoplasmic proteins or domains of proteins. If phoA is fused to a domain of a protein that is in the periplasm, it will result in alkaline phosphatase activity (yielding ax X-P+ colony). If phoA is fused to a domain of a protein that is in the cytoplasm, it will NOT result in alkaline phosphatase activity (yielding ax X-P- colony). For example, phoA fusions to the lacY protein, lactose permease, are shown below (Calamia, J., and C. Manoil. 1990. Proc. Natl. Acad. Sci. USA 87: 4937-4941).
Jacob, Lwoff, and Monod won a nobel prize for their work elucidating the regulation of the lac operon.
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Last modified April 25, 2000