Joany Chou

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.

Replication, establishment of latency, and induced reactivation of herpes simplex virus gamma 1 34.5 deletion mutants in rodent models.

Previous studies have shown that a gene mapping in the inverted repeats of the L component of herpes simplex virus, type 1 DNA, designated as gamma (1) 34.5, was dispensable for growth in cells in culture but that the deletion mutant (R3616) and a mutant containing a stop codon (R4009) in each copy of the gene were incapable of replicating in the central nervous systems (CNS) of mice. Restoration of the deleted sequences restored the wild type virus phenotype. We report here that the gamma (1) 34.5 mutant viruses (R3616 and R4009) replicated in the vaginal tract of two different strains of mice and guinea pig, although both viruses were shed at lower titer and for fewer days than the wild type and restored viruses. Both R3616 and R4009 failed to replicate or cause significant pathology in the cornea of Balb/C mice or following intranasal inoculation of Swiss Webster mice. Analyses of sensory trigeminal and dorsal root ganglia innervating the site of inoculation indicated that the incidence of establishment of latency or reactivation from latency by R3616 and R4009 viruses was significantly lower than that determined for mice infected with wild type or restored virus. Thus, selective deletion of gamma (1) 34.5 gene abolished the capacity of the virus to spread from peripheral mucosal sites to the CNS or replicate in the CNS, and diminished the capacity of the virus to replicate at mucosal sites and, subsequently, establish latency, or be able to be reactivated ex vivo. The results of our studies may have direct implications for the development of genetically engineered herpes simplex virus vaccines.

Isomerization of herpes simplex virus 1 genome: identification of the cis-acting and recombination sites within the domain of the a sequence

Previous studies have shown that the a sequence located at the termini and at the junction between the L and S components is the site-specific, cis-acting sequence mediating the inversions of herpes simplex virus 1 DNA. We constructed mutated a sequences, inserted them into the thymidine kinase gene, and recombined them into the L component of the viral genome. Deletion of Uc or Ub domains of the a sequence did not affect inversions, whereas the deletion of direct repeat #4 (DR4) drastically reduced their frequency. Deletion of both direct repeat #2 (DR2) and DR4 abolished inversions. Recombinational events leading to inversions appear to occur through DR2, and possibly DR4. These results complement previous studies showing that most of one DR1 sequence can also be dispensed with and are consistent with the hypothesis that DR4 and possibly DR2 are the cis-acting sites for the inversions mediated by the a sequence.

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.

Relationships among DNA sequences of the 1.3 kb EcoRI family of mouse DNA.

SummaryThe genome of the mouse (Mus musculus) contains a family of repeated DNA sequences defined by a 1.3 kb EcoRI fragment. Resqtriction maps of ten cloned fragments from this family have been determined. The fragments were of seven different types, based on the patterns of digestion obtained with AvaII, HindIII, and TaqI restriction enzymes. These seven unique sets of sequences fell into two classes, as defined by the position of a single HindIII site. Portions of fragments from each of the two classes were sequenced. Although certain regions of the repeat were highly conserved between classes, there was more intraspecific sequence divergence among the sequenced regions than has been observed for the short interspersedAlu family of repeated sequences sin mammals. Sequences of both HindIII classes were found to be present within the mouse X chromosome; we can conclude that both classes must also be present on other mouse chromosomes.

137 Enhanced tumor control of human glioblastoma multiforme xenografts with the concomitant use of radiotherapy and an attentuated herpes simplex-1 virus (ASTRO Research Fellowship)

Purpose: Glioblastoma Multifonne remains one of the most incurable of human tumors. The current treatment outcomes are dismal. There are several recent reports which suggest that some human glioblastoma xenografts implanted in the brains of athymic mice may be potentially cured with the use of an attenuated herpes simplex-l virus alone. We have chosen a replication competent, non-neurovimlent HSV-1 mutant, designated R3616 to determine whether there is an interactive cell killing and enhanced tumor control with radiotherapy in the treatment of a human glioblastoma xenograft.

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.