Controllable stability of DNA-containing polyelectrolyte complexes in water-salt solutions

Destruction of polyelectrolyte complexes (PECs) formed by DNA and synthetic polyamines of different structures was carried out by addition of low molecular weight electrolyte to PEC solution at different pHs. The dissociation was studied by the fluorescence quenching technique using the ability of cationic dye ethidium bromide to intercalate into free sites of DNA double helix followed by ignition of ethidium fluorescence. Structure of amine groups of the polycation was shown to be a decisive factor of PEC stability. PECs formed by polycations with quaternary amine groups, i.e., poly(N-alkyl-4-vinylpyridinium) bromides, poly(N, N-dimethyldiallylammonium) chloride, and ionene bromide, were pH independent and the least tolerant to destruction by the added salt. Primary amine groups of basic polypeptides poly-L-lysine hydrobromide and poly-L-arginine hydrochloride as well as synthetic polycation poly(vinyl-2-aminoethyl ether) provided the best stability of PECs in water-salt solutions under wide pH range. Moderate and pH-dependent stability was revealed for PECs included poly(N,N-dimethylaminoethylmethacrylate) with tertiary amine groups in the chain or branched poly(ethylenimine) with primary, secondary, and tertiary amine groups in the molecule. The data obtained appear to be the basis for design of DNA-containing PECs with given and controllable stability. The design may be accomplished not only by proper choice of polyamine of one or another type, but by using of tailor-made polycations with given composition of amine groups of different structure in the chain as well. Thus, quaternization of a part of tertiary amine groups of poly(N, N-dimethylaminoethylmethacrylate) resulted in expected decrease of stability of DNA-containing PECs in water-salt solutions. The destruction of PEC formed by random copolymer of 4-vinylpyridine and N-ethyl-4-vinylpyridinium bromide was pH sensitive and could be performed under pH and ionic strength closed to the physiological conditions. This result appears to be particularly promising for addressing DNA packed in PEC species to the target cell.     

Cloning of genetic material in Bacillus

Plasmid vectors capable for propagation of Bacillus subtilis DNA fragments containing riboflavin genes were constructed. Cloning of rib operon using pUB110 derivatives was performed in recE4 strain by using sequentional rescue of plasmids containing subfragments of the operon. Also, rib operon was cloned on the vectors containing DNA repeats. It was shown that the presence of direct and inverted repeats within plasmids allows to transform B. subtilis cells by monomers of plasmid DNA. Vectors that contained repeated sequences of DNA and ensured efficient cloning of genetic material in B. subtilis recipient cells were constructed. The use of streptococcal plasmid pSM19035 allowed to obtain vectors which were suitable for cloning large DNA fragments (6 MD and even more) in B. subtilis. A model of B. subtilis transformation by various types of plasmid DNA is presented. The model is in agreement with the general conception of chromosomal DNA transformation.     

Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage.

Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

Insertion of eukaryotic DNA into the Bacillus subtilis genome by means of a temperature-sensitive plasmid vector

A hybrid temperature-sensitive plasmid capable of integration into the Bacillus subtilis genome was constructed. By using this vector, we inserted a 3.2-kb fragment of eukaryotic DNA (wheat 'Chinese Spring') into the bacterial genome. The fragment of wheat DNA was stably retained and replicated as a part of the bacterial genome. The position of the integrated plasmid in the B. subtilis genome was mapped, as was the site in wheat DNA insert on plasmid at which the integration occurred.     

双启动子对重组溶源性枯草杆菌中外源蛋白表达的增强作用

探讨了双启动子对基于溶源性噬菌体构建的重组枯草杆菌中外源蛋白表达的影响。分别将不含或含有本身启动子的α-淀粉酶基因(来源于Bacillus amyloliquefaciens)和青霉素酰化酶基因(来源于Bacillus megaterium)克隆到溶源性枯草杆菌中,得到重组菌B.subtilisAMY1,B.subtilisAMY2,B.subtilisPA1以及B.subtilisPA2。由于同源重组,所克隆的片段整合到溶源性枯草杆菌中的噬菌体基因组上,并处于噬菌体强启动子的下游。在重组菌AMY1和PA1中,在热诱导的情况下外源基因的转录只受到噬菌体启动子的作用,而在重组菌AMY2和PA2中,在热诱导下外源基因的转录同时受到噬菌体启动子和基因本身所带启动子的作用。双启动子的应用使重组α-淀粉酶的表达量提高了133%,使重组青霉素酰化酶的表达量提高了113%。     

Transformation of Bacillus thuringiensis vegetative cells by electroporation

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.     

Construction of a Bacillus subtilis cloning vehicle with heterologous DNA sequence

A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI.     

Gene therapy of cystic fibrosis: the glycofection approach

Many cells express surface membrane lectins that selectively bind and carry glycoconjugates into intracellular endosomes; in addition, various intracellular membrane and soluble lectins act as shuttles between different compartments. On this basis, we developed glycosylated polycations, now called glycofectins (glycosylated polylysine and polyethyleneimine). Recently, we set up a simple way to transform oligosaccharides into glycosynthons suitable to substitute proteins or polymers. Glycofectins bind plasmid DNA leading to compact glycoplexes. Glycoplexes prepared with glycofectins were found to be much more active than naked plasmid to transfer genes to various types of cells including human airway epithelial and serous cells. The gene transfer efficiency was found to depend on the nature of the sugars borne by glycofectins. It appeared that the sugar-dependent efficiency was not only related to the uptake but also to the intracellular traffic of glycoplexes.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

Plasmid replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

Hybrid vectors for the cyanobacterium Anacystis nidulans R2, containing the plasmid pUB110 from Staphylococcus aureus

The recombinant vector plasmids were constructed having the DNA of pUB110 plasmid (4,5 kb, KmR) from Staphylococcus aureus inserted into the cryptic plasmids pANS (8 Kb) and pANL (48,5 kb) of cyanobacterium Anacystis nidulans R2. The hybrid plasmids transform cyanobacterial cells to Km-resistance with high efficiency. The plasmid pBS20, containing the complete sequence of pANS and pUB110 DNA, transforms Bacillus subtilis rec E4 protoplasts being, however, unstable in bacilli cells and disintegrates deriving a parent pUB110 plasmid.     

血红蛋白基因在枯草芽孢杆菌中的表达及其作用的研究

革兰氏阴性菌Ｖｉｔｒｅｏｓｃｉｌｌａ的血红蛋白与氧结合力强,能降低该菌需氧量。将该血红蛋白的结构基因（Ｖｇｂ）连接到枯草杆菌质粒ｐＡＫ４的β－内酰胺酶基因（ｂｌａ）启动子下（框架正确）,构建了重组质粒ｐＡＶ,并将此质粒先转化至枯草杆菌ＤＢ１０４,继而又转化到枯草杆菌碱性蛋白酶工程菌Ｇ３３１和产木聚糖酶枯草杆菌Ｂ５３。经酶切和电泳分析显示转化的质粒ＤＮＡ含有大小与Ｖｇｂ相同的电泳带,又经非放射性同位     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Properties ofthyP3-containing plasmids inBacillus subtilis

A series of hybrid plasmid molecules which contain both antibiotic resistance genes and the thyP3 gene of the Bacillus subtilis bacteriophage phi 3T have been constructed. Monomeric or restriction enzyme-cleaved plasmid DNA is capable of transforming competent cells to thymine prototrophy only. However, multimeric plasmid DNA can transform competent cells to both thymine prototrophy and antibiotic resistance. Cells which have been transformed to thymine prototrophy only do not contain extrachromosomal plasmid DNA but instead contain the thyP3 gene integrated into the host chromosome; the antibiotic resistance genes, however, do not become integrated into the chromosome. Although the thyP3-containing plasmids have extensive DNA sequence homology with the B. subtilis chromosome, they can be stably maintained, extrachromosomally, even in recE4+ hosts, in complex broth, and in the absence of antibiotics.