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GeneticsLactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: In vivo probe for transcriptional control sequences Communicated by A. Dale Kaiser, May 10, 1979 ABSTRACT The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage. This phage also carries a gene encoding resistance to ampicillin (Ap) [Mu(Ap, lac)J. After infection and upon establishment of lysogeny, the Mu(Ap, lac) genome can integrate into apparently random sites in the Escherichia coli chromosome. When integration occurs within a gene in the orientation of its transcription, the lactose structural genes are so situated that they become expressed solely from the promoter of that gene. Thus, expression of the lactose genes of Mu(Ap, lac) can be used as an assay for transcription of that gene and for functional and mutational studies of gene regulation.

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We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.

New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 ILI of a lysate containing 106 helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence onrT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation.The cloning of DNA sequences is an important step in many biological studies. Usually, cloning is done by isolating DNA, digesting it with restriction endonucleases, ligating it to an appropriately cut vector, and introducing it into a cell by transformation or transduction. As an alternative, transposable elements can carry out recombination reactions to translocate DNA onto plasmid or bacteriophage vectors to form gene clones (15,17,(34)(35)(36). The transposon bacteriophage Mu is a temperate phage that can infect Escherichia coli K-12 as well as other members of the family Enterobacteriaceae (for a recent review, see reference 34). Mu is especially suitable for in vivo cloning because it transposes hundreds of times as it replicates when derepressed for its lytic functions. Derepression can be achieved synchronously with temperature-sensitive alleles of the Mu repressor.We recently developed a more efficient in vivo cloning system by incorporating a plasmid replicon inside a mini-Mu element (17). Transposition of this mini-Mu replicon can occur on both sides of a particular gene to form a structure that can be encapsidated by the efficient Mu headful packaging mechanism (34). This structure can be introduced by phage infection into a Mu-lysogenic cell where recombination can occur between the Mu sequences to generate plasmid clones. This mini-Mu replicon element, Mu dII4042, can also form hybrid protein lac gene fusions which are useful in studies of gene structure and expression (9-11, 30).Here we describe the construction o...

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli 3-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid 3-galactosidase proteins. Conditions for transfection were varied to optimize the expression of 13-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli fgalactosidase and distinct from the major lysosomal "acid" fl-galactosidase. The fused preproinsulin-fl-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric P-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-,B-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed.Gene fusions utilizing the Escherichia coli lacZ structural gene provide an especially suitable method for studying gene expression and regulation (1). Its product, f3-galactosidase, can be assayed readily and does not require its NH2-terminal amino acids for enzymatic activity (2, 3). Fusions lacking NH2-terminal /-galactosidase gene codons can utilize not only the promoter and transcriptional regulatory elements but also the translational initiation and NH2-terminal amino acid codons from other genes joined to the lacZ gene and result in the formation of enzymatically active hybrid proteins. Such /3-galactosidase gene fusions have been used in bacteria and yeast for mapping and orienting genes and for locating their promoters, regulatory sites, coding regions, terminators, and other genetic elements (4-6). LacZ fusions also allow the use of lac operon genetic techniques as well as providing a convenient marker for subcellular localization (7) and purification (5, 8) which can be used for antibody induction (9) or amino acid sequence studies (10).In the present study we investigated the feasibility of using lacZ translational gene fusions to study gene expression in mammalian cells. The rat preproinsulin II and the simian virus 40 (SV40) gene controlling elements were fused to lacZ and were found to direct the expression of enzymatically active ,B-galactosidase proteins in COS-7 monkey kidney cells. MATERIALS AND METHODSPlasmid DNA. DNA cloning methods have been described elsewhere (11). Plasmids were propagated in E. coli strains M182