The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.     

Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs

A method is described for the isolation and purification of covalently closed circular (ccc) DNA from yeast (Saccharomyces cerevisiae). Spheroplasts are lysed at pH 12.45 which denatures linear but not ccc DNA. Next, the lysate is taken through a gentle high-salt-phenol extraction to remove single-stranded DNA. The ccc DNA, recovered by ethanol precipitation, can be further studied by agarose gel electrophoresis, can be cut with restriction endonucleases and can be used to transform Escherichia coli. This method efficiently purifies large (approx. 190 kb) and small (approx. 1.5 kb, TRP1-RI Circle) circular DNAs and thus has general applicability for isolation and purification of plasmids from yeast.     

A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae

A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).