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Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.
Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.
Numerous insertion mutations conferring resistance to antibiotics are available in Bacillus subtilis. However, many of them have been constructed in vitro by inserting genes from the Staphylococcus aureus plasmids, pC194 or pE194, conferring resistance to chloramphenicol (Cm) or erythromycin (Er). Others are insertions of the Enterococcus faecalis Tn917 transposon conferring resistance to Er. This paucity of resistance markers has been limiting the possibilities of constructing and studying mutants carrying two or more of these mutations in the past. We constructed plasmids which can be used to change the antibiotic resistance expressed by preexisting chromosomal insertions, through transformation and homologous recombination. These vectors replace the pre-existing resistance to Cm or Er with new resistances to neomycin (Nm), phleomycin (Pm), spectinomycin (Sp) or tetracycline (Tc).
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A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.
The copper resistance of a strain of Escherichia coli isolated from the effluent of a piggery where pigs were fed a diet supplemented with copper sulfate was controlled by a conjugative 78-megadalton plasmid designated pRJ1004. Plasmid pRJ1004 exhibited surface exclusion and incompatibility with standard plasmids belonging to incompatibility groups I1 and K. Sensitive strains of E. coli K-12 were unable to form colonies on nutrient agar containing more than 4 mM copper, whereas transconjugants which harbored pRJ1004 were able to form colonies on medium containing up to 20 mM copper.
A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a BglII-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.
A cryptic plasmid designated pSBO2 (3582 bp) was isolated from Streptococcus bovis JB1. The pSBO2 contained putative sites for a double-strand origin (dso), a small transcriptional repressor protein (Cop), countertranscribed RNAs (ctRNAs), and a replication protein (Rep), which were similar to those from pMV158 and pLS1, which were isolated from S. agalactiae, and pWVO1, isolated from Lactococcus lactis. The putative single-strand origin (sso) of pSBO2 was similar to pER341 and pST1, which were isolated from S. thermophilus. Recombinant plasmid designated pSBE2 was constructed to bind pECM184 vector and the DNA fragment containing sso, dso, Cop, ctRNAs, and Rep of pSBO2. When pSBE2 was introduced into S. bovis 12-U-1 and no8, the plasmids in the transformants had deleted the 160-bp fragment between sso and dso. This plasmid, designated pSBE2A, was capable of transforming Escherichia coli and S. bovis strains 12-U-1 and no8 on high frequency; therefore, pSBE2A is an effective shuttle vector.
The objective of this work was to further develop a tetracycline repressor (TetR) protein system that allows control of transgene expression. First, to circumvent the need for a binary approach, a single plasmid design was constructed and tested in tissue culture. To indirectly assay integrations that express the synthetic transcription factor (rtTA), a bicistronic gene was built which included an internal ribosome entry site (IRES) and a green fluorescent protein coding region (GFP) on the same expression cassette as the coding region of rtTA (pTetGREEN). This construct did not produce fluorescent colonies when stably integrated and provided minimal expression of GFP in the face of adequate expression of rtTA. The coding region for TetR was then altered by introducing 156 silent point mutations to simulate mammalian genes. Replacement of wild-type TetR gene (tetR) in pTetGREEN with 'mammalianized' tetR provided GFP expression. Adjustment of codon usage in the tetR region of rtTA nearly doubled the expression level of functional rtTA. To increase the number of rtTA expressing lines, the chicken egg-white lysozyme matrix attachment region (MAR) was introduced into the single plasmid design just upstream of the tetracycline operators (tetO). Inclusion of the MAR doubled the number of colonies that expressed rtTA (44% vs 88%). With the modifications described here, the number of lines that express rtTA and provide induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adjusting the base composition of the TetR coding region. The MAR also insulates the inducible gene from the promoter driving rtTA.
Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for tumour formation-the T(umour)-region and the Vir(ulence)-region. The T-region is transferred to plant cells by an unknown mechanism, and becomes stably integrated into the plant genome. The Vir-region has been identified by transposon mutagenesis, but the DNA of this region has never been detected in tumour lines. However, trans-complementation of Vir mutants indicates that genes of the Vir-region are functional in the bacterium. Moreover, the Vir- and T-regions can be physically separated in A. tumefaciens without loss of tumour-inducing capacity. Seven loci, designated virA-F and virO (refs 17, 20-22), have been identified in the Vir-region of the octopine Ti plasmid, but their functions are unknown. As virC mutants in the octopine-type plasmid pTiB6 are invariably avirulent in tests on various plant species, this gene seems to be essential for virulence and we are studying it in detail. We report here that the promoter of virC shows no detectable activity in A. tumefaciens and Escherichia coli K-12 grown in standard medium, but that its activity is induced by a plant product.
Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE DNA vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids encode a cassette consisting of the signal of premembrane (prM), prM, and envelope (E) coding regions of JE virus. One plasmid, designated pcJEME, is based on a commercial vector (pcDNA3), whereas the other plasmid, designated pNJEME, is based on a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine use. No differences were detected in the immunogenicity of these two plasmids in mice or swine. Swine immunized with the DNA vaccines at a dose of 100 to 450 microgram at an interval of 3 weeks developed neutralizing and hemagglutination-inhibitory (HAI) antibody titers of 1:40 to 1:160 at 1 week after the second immunization. However, swine administered two doses of a commercial JE vaccine (formalin-inactivated virus preparation; JEVAX-A) developed low (1:10) or undetectable antibody responses after their boost. Interestingly, serum antibody titers elicited by DNA vaccines in swine were higher than those detected in mice. Eight days after boosting with viral antigen (JEVAX-A) to detect an anamnestic response, swine immunized two times with the DNA vaccine showed a >100-fold elevation in HAI titer, indicating a strong recall of antibody response. Swine maintained detectable levels of HAI antibody for at least 245 days after two immunizations with a DNA vaccine. These results indicate that these DNA vaccines are able to induce virus-specific memory B cells and long-lasting antibodies in swine, which were of higher levels than those obtained with a commercial formalin-inactivated JE vaccine.
Two Escherichia coli strains isolated from urinary tract infections were resistant to streptomycin, kanamycin, neomycin, tetracycline and sulphonamides. The strains also produced colicins B and M. The resistance to streptomycin, kanamycin and neomycin and the ability to produce colicins B and M could be transferred to an E. coli K12 recipient. Resistance and colicinogeny markers were transferred together by conjugation, and did not segregate even after interrupted mating or phage P1-mediated transduction. Hence, the drug-resistance and colicinogeny markers were carried by the same plasmid, designated as R.ColBM plasmid. The two R.ColBM plasmids were Fi+ and produced an F-like pilus. They belonged to the IncFIII incompatibility group, being thus the first R plasmids identified in this group. The two plasmids were isolated and their molecular sizes were determined by electrophoresis in agarose gels and by contour length measurements. Both methods showed that both plasmids were about 52 megadaltons.
The gene (entD) encoding staphylococcal enterotoxin D (SED) has been located on a 27.6-kilobase penicillinase plasmid designated pIB485. This plasmid was present in all SED-producing strains tested. The entD gene was cloned on a 2.0-kilobase DNA fragment and was expressed in Escherichia coli. Sequence analysis of this fragment revealed an open reading frame that encoded a 258-amino-acid protein that possessed a 30-amino-acid signal peptide. The 228-amino-acid mature polypeptide had a molecular weight of 26,360 and contained a high degree of sequence similarity to the other staphylococcal enterotoxins. S1 nuclease mapping showed that transcription of entD was initiated 266 nucleotides upstream from the translation start codon. The entD gene was also shown to be activated by the staphylococcal regulatory element known as agr.
To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.
Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10(6) transformants per microg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.
The requirement for both plasmid and chromosomal genes in the biosynthesis of Shigella dysenteriae 1 lipopolysaccharide O antigen was demonstrated in Escherichia coli-Shigella hybrids. A 6-megadalton S. dysenteriae 1 plasmid, designated pWR23, was phenotypically tagged with the Tn3 ampicillin-resistance transposon. The tagged plasmid, designated pWR24, was transferred by transformation or conjugal mobilization to a rough E. coli K-12 recipient. Although the resultant hybrids were agglutinated in S. dysenteriae 1 antiserum, they did not remove all of the anti-Shiga agglutinins in absorption experiments. Modified lipid A core structure was detected in these hybrids, but Shiga O antigen was not expressed. When the his+ locus of the S. dysenteriae 1 chromosome was transferred by transduction to E. coli K-12 containing pWR24, complete Shiga O antigen was expressed. Lipopolysaccharide extracted from these hybrids was indistinguishable chemically, electrophoretically, and serologically from native S. dysenteriae 1 lipopolysaccharide.
Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.
A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.