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.     

Denaturation maps of DNA: experimental and theoretical maps of phiX174 DNA.

A formaldehyde denaturation map of the replicative form of phiX174 DNA is obtained. The RFI DNA was converted into a linear state by restriction endonuclease pst I which introduces into this DNA a single double-stranded break. The map has four clear-cut peaks. Their positions excellently correlate with the peak positions on the map of equilibrium denaturation theoretically obtained earlier from the known nucleotide sequence of phiX174 DNA. The sequence is also used for a calculation of the maps of smoothed AT-content. The maxima on these maps correlate well with the peaks on the denaturation maps. To reveal the causes of a good correlation between the experimental formaldehyde and theoretical equilibrium denaturation maps, the theoretical formaldehyde denaturation maps are calculated for different conditions (temperature, formaldehyde concentration) using the detailed theory of DNA interaction with formaldehyde developed earlier.     

Denaturation map of polyoma DNA.

A denaturation map of polyoma DNA cleaved by Eco R1 to form linear molecules was established by electron microscopy. Partial denaturation, under the same conditions, of fragments obtained by Haemophilus influenzae restriction enzymes allowed us to align the denaturation map with the already established physical map of polyoma DNA (Griffin et al., 1974).     

Construction and characteristics of mini-Mu phages

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Mini-muduction: A new mode of gene transfer mediated by mini-Mu

Summary We compared the transducing properties of Mucts62 and Mucts62/mini-Mu lysates, using Mu immune and non immune Rec⁺ and recA recipient strains. The Mu/mini-Mu lysates transduced all bacterial markers tested 10 times more efficiently than the Mucts62 lysates in Rec⁺ recipients. Most of the transductants obtained after infection with the Mu/mini-Mu lysates result from the substitution of the mutated gene of the recipient by the wild type allele from the donor, most probably carried on the gigantic variable end linked to the mini-Mu genome.Moreover the Mu/mini-Mu lysates gave a new type of Rec-independent transduction that we called mini-muduction. Mini-muduction requires the activity of Mu gene A and provides transductants which carry the transduced marker surrounded by two mini-Mu genomes similarly oriented, and inserted at random location in the recipient chromosome. The mini-Mu/transduced DNA/mini-Mu structures are able to transpose spontaneously, for instance into a transmissible plasmid, in the presence of Mu gene A product.     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis. Denaturation mapping by electron microscopy.

Electronmicroscopic observation of the denaturation pattern of 130 partially denaturated linear mitochondrial DNA molecules from Saccharomyces carlsbergensis was used to investigate the distribution of AT-rich sequences within the mitochondrial genome. The molecules were observed after heating to 43 degrees C in the presence of 12% formaldehyde. These conditions resulted in an average denaturation per molecule of 21%. The average length of the molecules was 10 mum, and a few molecules had a length corresponding to the size of the complete genome. The undenaturated regions varied in length from 0.1 to 5.0 mum with denaturated regions of length 0.02 to 0.1 mum in between. A denaturation map was constructed by use of one of the long molecules (28.7 mum) as a master molecule for positioning of all other molecules. This map shows distinct regions corresponding to the position of easily denaturated sequences in the mitochondrial DNA. These sequences which presumably correspond to the very AT-rich regions, known to exist in the yeast mitochondrial DNA, were found at intervals of about 0.5 - 3 mum on the map.     

A cos-mini-Mu vector for in vivo DNA cloning

Construction of a mini-Mu plasmid vector containing a cosmid replicon is described. Upon derepression of mini-Mu transposition, bacterial DNA sequences can be flanked by the integrated mini-Mu. These sequences can then be packaged into lambda heads by superinfection with a lambda helper phage. Cosmid clones carrying particular bacterial genes can be recovered by selection after infection of appropriate strains with the cosmid transducing lambda lysate. We report here the successful in vivo cloning of several Escherichia coli genes using the transposoncosmid vector.     

A map of the sites on bacteriophage PM2 DNA for the restriction endonucleases HindIII and HpaII

The map of the seven sites for the restriction endonuclease HindIII³ and the single site for endo R.HpaII on PM2 DNA was determined. This map was oriented with respect to the denaturation map of this DNA (Brack et al., 1975) by partial denaturation mapping of the fragments. A new method for localizing restriction fragments by DNA-DNA hybridization and electron microscopy is described.     

Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae

Summary The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a T_m of 80.6°C and the second phase has a T_m of 87.3°C, corresponding to GC contents of 28 and 44%, respectively. The T_m of the nonribosomal nuclear DNA, called DNA, is 85.7°C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6×10⁶ dalton rDNA SmaI restriction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions.A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.     

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].     

Physical map of the DNA of bacteriophage phiB

A physical map of DNA phi B bacteriophage was constructed. Using the data of denaturation maps and heteroduplex analysis the positions of 10 (from 11) peaks were estimated. These peaks belong to DNA regions obtained after hydrolysis of phi B DNA by EcoRI endonuclease. This map is orientated to the end of the DNA molecule, which first entered the head, during phage maturation. The disposition of AT-enriched regions of the DNA molecule were shown.     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA.

Interactions between the Mu A and Mu B proteins are important in the early steps of the in vitro transposition of a mini-Mu plasmid. We have examined these interactions by assaying Mu B stimulation of Mu A-mediated strand cleavage and strand transfer reactions. We have previously shown that in the presence of ATP the Mu B protein can stimulate the Mu A-directed cleavage reaction of mini-Mu plasmids carrying a terminal base pair mutation (Surette, M.G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266, 3118-3124). Here we demonstrate that in the absence of a non-Mu DNA target molecule the Mu B protein stimulates intramolecular integration of a mini-Mu in an ATP-dependent fashion. Furthermore, modification of the Mu B protein with N-ethylmaleimide severely compromises the ability of B to form a stable complex with DNA; however, the modified protein stimulates the strand cleavage and intramolecular strand transfer reactions as efficiently as the untreated protein. These results indicate that the Mu B protein is capable of stimulating the Mu A protein through direct interaction in the absence of stable Mu B-DNA complex formation. Our results increase the spectrum of Mu B protein activities and uncouple the stimulatory properties of the Mu B protein from stable DNA binding but not the ATP cofactor requirement.     

Transposition of mini-Mu containing only one of the ends of bacteriophage Mu.

Transposition of mini-Mu containing only one of the ends of bacteriophage Mu was studied. Transposition was observed when plasmids containing this mini-Mu were used as the donor in a mating-out assay with the F factor POX38, which lacks all known transposable elements, as the target. Analysis of the plasmids isolated from the recipient strain showed that in most cases the mini-Mu containing plasmid and POX38 were fused and that a part of the plasmid had been duplicated, indicating the formation of co-integrates. To obtain the DNA sequences of the junctions between donor and recipient DNA, an F factor was constructed that made it possible to analyse these junctions directly. The results showed that several sequences can be used as an alternative end in transposition and that these alternative ends show homology with the consensus for a strong A binding site. Moreover, the first base pair at the junction was always a (TA) base pair. This base pair is situated at exactly the same position with respect to the sequence which has homology with the consensus for a strong A binding site as in the ends of Mu.     

Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction.

The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated. Insertion of a mini-Mu molecule into certain sites in pUC19 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition. Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type 1-deficient mini-Mu molecules. Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends. This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction. Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.     

DNA of the Streptomyces phage SH10: Binding sites for Escherichia coli RNA polymerase and denaturation map

Summary Escherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.     

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.