Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage P1 cloning vector

A bacteriophage P1 cloning system that permits the isolation and amplification of cloned DNA fragments as large as 100 kbp was described previously. We have now utilized a similar system to generate a 50,000-member human DNA library with DNA inserts ranging in size from 75 to 100 kbp. Two major obstacles were overcome in constructing the library. The first concerned the mcrAB restriction system of Escherichia coli, which degrades DNA containing MeC and interferes with the recovery of cloned human DNA inserts. In the P1 cloning system, the effect of the Mcr restriction activity is to decrease recovery of cloned inserts by about 35-fold when the activity is in the host cell line and by about 3-fold when the activity is in the cells used to prepare the packaging extract. To circumvent this problem we inactivated, by mutation, the McrAB proteins in both components of the cloning system. The second obstacle concerned the preferential cloning of small DNA fragments from a population of fragments ranging in size from 20 to 100 kbp. To deal with this problem we first modified the P1 lysogen used to prepare the in vitro head-tail packaging extract so that it would produce 12 times as many large P1 heads (head capacity about 110 kbp) as small P1 heads (head capacity about 45 kbp). We then restructured the P1 cloning vector so that it could be used to produce vector "arm" fragments that could be ligated to insert DNA at only one end. This prevented the formation of long concatamers consisting of alternating units of vector and insert DNA and prohibited the packaging of small inserts in large phage heads. Using the insert-biased large head extract, the arms vector, and size-selected human DNA fragments, we showed that as much as 90% of recovered transformants contained inserts in the desired high molecular weight range.     

The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome

Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.     

A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments

ABSTRACT: BACKGROUND: Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites. FINDINGS: The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps. CONCLUSIONS: Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs.     

Agarose gel electrophoretic evidence for domains of nuclear DNA linked with bonds cleavable with sulfhydryl molecules

Complexes of intact nuclear DNA with proteins undissociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage lambda DNA) into smaller-Mr forms. Following limited restriction endonuclease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60-110 kbp range. These 60-110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.     

Conversion of bacteriophage fd into an efficient single-stranded DNA vector system

Summary Single-stranded DNA vectors were constructed in vitro by insertion of various DNA fragments into the Intergenic Region of the single-stranded DNA phage fd. These inserts introduce into the phage genome unique cleavage sites for restriction nucleases which are suited for sticky joining in cloning experiments. Since these sites are usually located within genes coding for antibiotic resistance, inactivation of a resistance gene by insertion can be used as a marker for the successful cloning of a DNA fragment. Resistance genes also allow to select for recombinant DNA phages and to minimize the loss of DNA inserts which otherwise becomes significant above an insert size of about one kb. Cloning of several DNA fragments is described and strand separation of double-stranded DNA fragments by means of cloning into fd DNA is given as an example for application of single-stranded DNA vectors.Abbreviations Ap ampicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - kb, kbp a unit length equivalent to 1000 bases, respectively 1000 base pairs - wt wild type     

A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli

A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained. The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29. The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both. Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form. DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.     

A phage T4 in vitro packaging system for cloning long DNA molecules.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.     

Periodicity of DNA folding in higher order chromatin structures.

Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.     

An efficient synthetic primer for the M13 cloning dideoxy sequencing system

The deoxytetradecamer d(AAAACGACGGCCAG) has been shown to be an excellent universal primer for sequence determination of DNA cloned into the bacteriophage M13 mp7, mp8, and mp9 series. This new primer offers several advantages over others currently available and it has been used to define the cloning of Hinf I fragments of bacteriophage S13 DNA into the Eco RI site of M13 mp7, utilizing the homologous complementary base pairing of the two restriction sites. Of the four possible sequence derivatives of the Hinf I GANTC recognition site, only those corresponding to GAATC and GATTC have been found at cloning sites in chimeras.     

Temperate SM phage from Pseudomonas aeruginosa as a vector for cloning genetic information]

The recombinant bacteriophages with the genomes containing the DNA fragments of bacteria Erwinia chrysanthemi, including the pectatelyase gene, were constructed on the base of Pseudomonas aeruginosa temperate bacteriophage SM. The gene transferred into Pseudomonas aeruginosa PAO1 cells by transfection is expressed in the new bacterial host. The restriction maps of the recombinant bacteriophages are constructed and the position of an insert is defined. Bacteriophage SM was found to be capable of reproducing in Pseudomonas aeruginosa PAO1 cells when its DNA was shortened to 88% or increased to 111% of the normal genome length. Except for bacteriophage SM, the recombinant bacteriophage SM-2 with an unique restriction endonuclease site for XbaI can also be used as a vector for cloning. Bacteriophage SM capacity in cloning of heterological DNA at HindIII sites is not less than 8 Md, capacity of bacteriophage SM-2 is not less than 5 and 8 Md at XbaI and HindIII sites respectively.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

Cloning fragments of bacteriophage T5 DNA, determining expression of phage-dependent ligase

Cloning vèctor lambda gt-p MB9 has been used for cloning of DNA fragments of bacteriophage T5 produced by EcoR*I activity. One clone contains a DNA fragment of 2.2 Md which has been mapped at 67-71% on the physical map of the genome. Functional studies have shown that bacteriophage lambda gt-T5 can grow on E. coli lights 7. Infection of this E.coli strain with phage lambda gt-T5 induces DNA-ligase activity which has been previously observed in E. Coli infected with bacteriophage T5.     

Characterizing seamless ligation cloning extract for synthetic biological applications

Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular and synthetic biology projects.     

Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.     

An Arrayed Bacteriophage P1 Genomic Library of Pneumocystis carinii

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.     

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.     

A PCR-free cloning method for the targeted φ80 Int-mediated integration of any long DNA fragment, bracketed with meganuclease recognition sites, into the Escherichia coli chromosome

The genetic manipulation of cells is the most promising strategy for designing microorganisms with desired traits. The most widely used approaches for integrating specific DNA-fragments into the Escherichia coli genome are based on bacteriophage site-specific and Red/ET-mediated homologous recombination systems. Specifically, the recently developed Dual In/Out integration strategy enables the integration of DNA fragments directly into specific chromosomal loci (Minaeva et al., 2008). To develop this strategy further, we designed a method for the precise cloning of any long DNA fragments from the E. coli chromosome and their targeted insertion into the genome that does not require PCR. In this method, the region of interest is flanked by I-SceI rare-cutting restriction sites, and the I-SceI-bracketed region is cloned into the unique I-SceI site of an integrative plasmid vector that then enables its targeted insertion into the E. coli chromosome via bacteriophage φ80 Int-mediated specialized recombination. This approach allows any long specific DNA fragment from the E. coli genome to be cloned without a PCR amplification step and reproducibly inserted into any chosen chromosomal locus. The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry.     

Characterization of bacteriophage T3 DNA ligase

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.