Biomedical subjects

P Sugiono

Publications and source records attributed to P Sugiono.

4 recordsLinked to original sources

PubMed1989

Genetic selection for genes encoding sequence-specific DNA-binding proteins.

We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins. The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins. Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins. Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA. Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein.

Animals↗

PubMed1988

Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages.

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.

Chromosomes, Bacterial↗

PubMed1987

DNA specificity determinants of Escherichia coli tryptophan repressor binding.

We have analyzed the sequence-specific interaction between the Escherichia coli tryptophan (Trp) repressor and its operator using challenge phage vectors. These phages, derivatives of Salmonella phage P22 that have substitutions of synthetic, symmetric trp operators for the P22 mnt operator, provide a genetic assay for DNA binding in vivo. Phages carrying operators that retain the determinants of Trp repressor binding efficiently lysogenize cells producing repressor; in contrast, phages with operators missing critical determinants kill such hosts. The binding determinants revealed by this assay corroborate a simple docking model for the Trp repressor-operator interaction postulated from the repressor crystal structure, and account for both the specificity of repressor binding and the ability of Trp repressor to recognize multiple, tandem DNA sites.

Bacterial Proteins↗

PubMed1986

General selection for specific DNA-binding activities.

We present a general strategy for the selection of bacterial clones that express DNA-binding activities corresponding to particular DNA recognition sites. The selection uses a "challenge phage" vector, P22 Kn9 arc-amH1605, into which is substituted a synthetic DNA-binding site for a site that controls transcription of the P22 antirepressor (ant) gene. Constitutive synthesis of antirepressor channels a challenge phage into lytic development and efficiently kills an infected host, unless the substituted site is bound by a specific protein; in this case, the challenge phage prefers lysogenic development, and the host survives and acquires an antibiotic-resistance phenotype. Infections with challenge phages carrying the E. coli Lac operator, phage lambda OL1 operator, or synthetic, "idealized" E. coli Trp and Tn10 Tet operators select clones that express each of the corresponding binding activities. The use of challenge phage vectors may be extended to select clones that express eukaryotic DNA-binding activities.

Base Sequence↗