[33] Mud(Ap, lac)-generated fusions in studies of gene expression

One of the most powerful tools to be developed in recent years for use in genetic studies of Escherichia coli and related bacteria is the operon fusion vector, Mu c ts dl(ApR, lac) also referred to as Mud(Ap, lac). This bacteriophage, constructed by Casadaban and Cohen, allows lac operon fusions to virtually any genetic locus to be obtained in a single step. Previously, the construction of operon fusions required a somewhat laborious two-step procedure. The introduction of Mud(Ap, lac) has not only facilitated the construction of lac operon fusions to particular genes of interest, but has also permitted the conceptually new experimental strategy of screening for genes that are members of a common regulatory network without their functions being known in advance. The basic elements of the Mud(Ap, lac) bacteriophage are outlined in this chapter. The Mud(Ap, lac) bacteriophage creates operon fusions, not gene fusions; as the lac genes have a complete set of translational signals, normal B-galactosidase is synthesized. Second, insertion of Mud(Ap, lac) within a gene usually leads to inactivation of that gene and, as a consequence of the polarity exhibited by Mu, also prevents the expression of downstream genes within the same transcriptional unit.