Expression of fX174 bacteriophage gpA and gpA* replication proteins: Internal promoters and toxic gene products. Solvander, O & Love, C.A. Eskitis Institue for Cell and Molecular Therapies & School of Biomolecular and Physical Sciences, Griffith University, Nathan, Qld, Australia. The faithful replication of DNA is pivotal for the survival of all organisms. However, small bacteriophage such as fX174 relies on its E. coli host for almost all its replication proteins. The only encoded proteins implicated in the replicating bacteriophage DNA are gene protein A (gpA) and gene protein A* (gpA*), and these proteins are involved in initiation of replication and shut-down of host DNA replication, respectively. Both proteins are encoded by the same gene and gpA* is expressed from an internal, in-frame ATG start codon. The resulting gpA* represents the C-terminal 341 residues of gpA and was reportedly translated from a polycistronic mRNA transcribed from a promoter located upstream of gene A. Attempts to clone gene A to produce quantities of gpA for structural and functional characterisation have been unsuccessful. Investigation of the 5' upstream region of gene A* revealed the presence of a putative promoter region, and if functional may allow the constitutive expression of gpA* during cloning, leading to the shut-down of host DNA replication which is lethal. We have shown that the placement of this putative region upstream of the luc gene resulted in the expression of luciferase confirming that the region acts as a promoter. The expression levels obtained from this promoter were twice that obtained using the lac promoter, and when inserted in the reverse orientation relative to the luc gene the expression levels were reduced by 98.6%.