Malcolm J. Casadaban

Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

Abstract Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription. Expression of the lac genes in the absence of the inducer arabinose indicates that transcription originates within the inserted fragment; non-expression of lac with arabinose present indicates that transcription is terminated by the fragment. Using different cloning vectors, DNA fragments generated by a wide variety of restriction endonucleases can be inserted between ara and lac. This procedure has been used to identify and isolate endonuclease-generated DNA fragments from theEscherichia coli chromosome, various R plasmids, bacteriophage T5 and Drosophila melanogaster that contain nucleotide sequences capable of functioning as promoters in E.coli. A characteristic level of lac expression is determined by the amount of transcription that proceeds to the lac genes from a promoter located within each fragment. The effects of genetic regulatory mechanisms acting on a promoter can be assayed by alterations in the level of lac expression. These cloning vectors were also used to bring structural genes located within an inserted DNA fragment under the control of the ara promoter. Insertion of HindIII endonuclease-generated fragments carrying the tetracycline-resistance determinant of pSC101 or the sulfonamide-resistance determinant of the R6-5 plasmid into such vectors resulted in arabinose-induced resistance to tetracycline or sulfonamide.

Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast

Publisher Summary Gene fusions can be constructed either in vivo , using spontaneous nonhomologous recombination or semi-site specific transposon recombination, or in vitro , with recombinant DNA technology. This chapter describes in vitro methods and lists some recently developed β -galactosidase gene fusion vectors. With these methods, gene-controlling elements from any source can be fused to the β -galactosidase structural gene and examined in the prokaryote bacterium Escherichia coli or the lower eukaryote yeast Saccharomyces cerevisiae. β -galactosidase gene fusions can be constructed both with transcription initiation control signals and with transcription plus translation initiation control signals. The β -galactosidase gene is convenient for making translational fusions because it is possible to remove its translation initiation region along with up to at least the first 27 amino acid codons without affecting β -galactosidase enzymic activity. The focus here is on these transcription-translation fusions because they provide all the gene initiation signals from the other gene. β -Galactosidase expression from a gene fusion can be used not only to measure gene expression and regulation, but also to isolate mutations and additional gene fusions.

New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacA gene sequences encoding an enzymatically active carboxy-terminal portion of β-galactosidase

A new class of plasmid cloning vectors has been constructed with cleavage sites in a variety of translational reading phases of the promotorless lacZ gene. Fused hybrid proteins can be produced by these vectors by cloning DNA fragments containing the promoter, translation initiation site, and the amino terminal portion of a gene, all with proper orientation, into the correct translational reading frame of the lacZ gene. Enzymatically active hybrid-beta-galactosidase proteins are formed, which have amino-terminal amino acids encoded by the cloned gene segment. Another class of these vectors retains an active lac promoter and lacZ translation-initiation region, which can direct hybrid protein synthesis from DNA fragments that do not have gene initiation regions. These vectors allow transcription from the lacZ initiation region to proceed across, or to stop and restart within, an inserted fragment into the essential part of the beta-galactosidase gene. Also described is a small lacZ gene fragment (cartridge), without a plasmid replicon and without any other lac genes, which can be inserted directly into other genes to form hybrid protein fusions. Polyrestriction site sequences were easily moved into some of these vectors by incorporating drug-resistance genes that serve as markers for the selection and detection of these sequences; those markers can be easily removed afterwards.

Overproduction of the Tn3 transposition protein and its role in DNA transposition

Five single base pair mutations that increase expression of the tnpA (transposase) gene of the Tn3 transposon approximately 30-fold, but which still allow the gene to be regulated, have been isolated by using a generally applicable procedure that involves distally linked lac gene fusions. The mutations, which are all located in a region controlling initiation of translation of the tnpA gene, do not affect normal repression of tnpA by the tnpR gene product, and yield up to a 9000-fold increase in tnpA protein production when combined with a tnpR mutation and placed on a high copy number plasmid. The mutation yielding the highest expression level was separated from the fused lac gene segment by homologous recombination and was found to increase the rate of transposition without altering the nature of the transposition product; in cells defective in both the E. coli recA gene and the tnpR gene of tn3, cointegrate transposition-intermediate structures occur with the overproducing--as well as with the wild-type--tnpA gene. In the presence of a functional Tn3 tnpR gene or the related transposon delta gamma, such cointegrate structures are resolved into the final products of transposition.

Genome mapping and protein coding region identification using bacteriophage Mu

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35-kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.

In vivo DNA cloning with a mini-Mu replicon cosmid and a helper lambda phage.

A mini-Mu bacteriophage, containing the cohesive-end packaging site (cos) from a lambda-phi 80 hybrid phage, a high-copy-number plasmid replicon, and a kanamycin-resistance gene for independent selection, was constructed to clone genes in vivo. This mini-Mu element can be derepressed to transpose at a high frequency. DNA segments that become flanked by copies of this mini-Mu element in the same orientation can be packaged by a helper lambda phage. The resulting lambda lysate can be used to infect recipient cells where the injected DNA can circularize by annealing at the cos termini. Drug-resistant transductants obtained carry the mini-Mu-replicon cosmid element with inserts of different nucleotide sequences. These are analogous to recombinant DNA clones generated in vitro with restriction endonuclease cutting and ligase joining reactions replaced by the Mu transposition process. Clones of particular genes were isolated by their ability to complement specific mutations. Both recA+ and recA- recipient cells can be used with equal efficiency. Clones obtained with a helper lambda phage require the presence of the cos site in the mini-Mu replicon. They carry larger inserts than those isolated with the same mini-Mu element and Mu as a helper phage. The mini-Mu replicon-cosmid bacteriophage contains a lac-gene fusing segment for isolating fusions of lac operon DNA to gene control regions in the cloned sequences. Independent clones of a particular gene can be used to prepare a restriction map of the gene and its flanking regions.

Hybrid protein thymidine kinase gene fusions: plasmid vectors for the study of transcription and translation initiation signals

The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.

23 – β-Galactosidase Gene Fusions for Analyzing Gene Expression in Escherichia coli and Yeast

Publisher Summary This chapter focuses on β-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. The β-galactosidase structural gene lacZ can be fused to the promoter and controlling elements of other genes as a way to provide an enzymic marker for gene expression. β-Galactosidase gene fusions can be constructed both with transcription initiation control signals and with transcription plus translation initiation control signals. β-Galactosidase expression from a gene fusion can be used not only to measure gene expression and regulation, but also to isolate mutations and additional gene fusions. Higher levels of β-galactosidase are indicated if lactose can be utilized for growth on minimal media. β-Galactosidase fusions can be used in more species for which DNA transformation is possible, such as in gram-positive bacteria Bacillus subtilis and Streptomyces and in higher eukaryotes, such as plants and mammalian tissue culture cells.