Novel shuttle vectors for improved streptokinase expression in streptococci and bacterial L-forms

Novel shuttle vectors of small size and increased copy number capable of replication in Escherichia coli, L-forms of Proteus mirabilis, and streptococci were constructed from a streptococcal erythromycin-resistant plasmid and an Escherichia coli phasmid. The streptokinase gene, skc, was inserted into one of them, and skc expression was studied in Streptococcus sanguis, Streptococcus lactis, and in an L-form strain (LVI) of Proteus mirabilis. The new streptokinase shuttle plasmid, pMLS10 (7.3 kb), specified higher Skc yields in all hosts when compared to pSM752 constructed previously. In particular Proteus mirabilis LVI(pMLS10) proved to be the most productive host, exhibiting complete secretion of the active protein at yields as high as 24000 unit per ml.     

A shuttle system for transfer of YACs between yeast and mammalian cells.

The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.     

Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences

We describe the construction and properties of pEMBLY plasmids. They belong to a new family of yeast shuttle vectors which are derived from plasmid vector pEMBL9 and offer the following improvement: relatively small size; large number of cloning sites; screening for insert-containing plasmids on indicator plates; different combinations of genes which complement auxotrophic deficiencies and sequences that support DNA replication in Saccharomyces cerevisiae; and ability to isolate the plasmid DNA in single-stranded (ss) form. The yeast S. cerevisiae can be efficiently transformed by these plasmids in both the ss and double-stranded (ds) forms. Finally, the presence of the phage f1 intergenic region allows one to obtain the cloned sequences in the ss form upon infection with the wild-type ss phage [Dotto et al., Virology 114 (1981) 463-473].     

Family of shuttle vectors for ruminal Bacteroides

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 10(5) transformants/microg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11-0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0.53% per generation.     

Lambda phage shuttle vectors for analysis of mutations in mammalian cells in culture and in transgenic mice

Foreign DNA sequences contained in lambda bacteriophage genomes integrated in mammalian DNA can be efficiently rescued into infectious phage particles by treatment of the mammalian DNA with lambda-packaging extracts prepared in E. coli. This system provides for rapid, non-selective recovery of stably integrated, chromosomal sequences into lambda phage for subsequent analysis in bacterial systems. Since rescue is prior to selection, mutations can be recovered from intact animals made transgenic for the phage-target gene sequences. Such approaches allow study of physiologically relevant aspects of mammalian mutagenesis at the molecular level.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.     

Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast. We show the production of a helminth antigen as an example of the vector application.     

Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e. g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.     

Automatic elimination of unnecessary bacterial sequences from yeast vectors.

Most vectors for Saccharomyces cerevisiae are shuttle vectors which can be both propagated and selected in Escherichia coli. The DNA segments, however, which are required for propagation in E. coli are unnecessary and moreover toxic in S. cerevisiae. To delete these harmful DNA fragments from the vector after it is introduced into S. cerevisiae cells, we propose a specific gene conversion mechanism of a yeast plasmid, pSR1. Plasmid pSR1 has a pair of inverted repeats (IRs) that divides the plasmid molecule into two unique regions. Intramolecular recombination frequently occurs at a pair of specific recombination sites in IRs catalyzed by recombinase R, encoded by a pSR1 plasmid gene. This R-mediated recombination is often accompanied by gene conversion in IRs. Thus, a 2.1-kb pBR322 sequence for the E. coli host ligated into one of the IRs of a composite plasmid was automatically and effectively eliminated when the plasmid was introduced into S. cerevisiae cells.     

New shuttle vectors for Actinobacillus actinomycetemcomitans and Escherichia coli

A series of shuttle vectors for Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was developed. These vectors carry a multiple cloning site, the lacZα reporter gene, the replication origin for Aa derived from pVT736-1 and a replicon for Ec originated from pUC, P15A or pSC101. With these vectors, cloning and expression can be effectively performed in Aa and Ec.     

Construction of shuttle vectors useful for transforming Clostridium acetobutylicum

The symbiotic plasmid (pSym) DNA present in bacteroids of strain RCR1001 of Rhizobium leguminosarum biovar viceae has been compared qualitatively and quantitatively with that present in free living bacteria by hybridization experiments with appropriate probes. A decrease in the relative amount of pSym DNA was observed in bacteroids as compared to bacteria. No rearrangements of the symbiotically expressed pSym borne genes were detected in bacteroids.     

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).     

Novel streptococcal-integration shuttle vectors for gene cloning and inactivation.

Seven new streptococcal integration shuttle vectors have been constructed which contain different antibiotic-resistance-encoding genes capable of expression in both Streptococcus sp. and Escherichia coli. These plasmids can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The size, antibiotic resistance, and number of unique restriction sites available for cloning for each plasmid are as follows: pSF141 (7.6 kb, CmR and KmR, 7 sites), pSF143 (5.7 kb, TcR, 6 sites), pSF148 (7.3 kb, CmR and SpR, 7 sites), pDL285 (3.4 kb, KmR, 3 sites), pDL286 (3.1 kb, SpR, 4 sites), pSF151 (3.5 kb, KmR, 10 sites), pSF152 (3.2 kb, SpR, 9 sites). If these plasmids carry a fragment of streptococcal DNA they can specifically integrate into the chromosome via Campbell-like, homologous recombination. Therefore, they should be useful for gene inactivation, cloning, chromosomal walking, or linkage analysis in streptococci. The availability of these integration plasmids resistant to different antibiotics, along with the previously described plasmid, pVA891 (ErR), should also allow the construction of mutants possessing multiple insertionally inactivated genes useful for a variety of genetic studies.     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.     

Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.