Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

Are single-stranded circles intermediates in plasmid DNA replication?

Plasmid pC194 exists as circular double-stranded and single-stranded DNA in Bacillus subtilis and Staphylococcus aureus. We report here that the plasmid pHV33, composed of pBR322 and pC194, exists as double- and single-stranded DNA in Escherichia coli, provided that the replication functions of pC194 are intact. Single-stranded pHV33 DNA is converted to double-stranded DNA by complementary strand synthesis probably initiated at rriB, a primosome assembly site present on pBR322. The efficiency of complementary strand synthesis affects the double-stranded copy number, which suggests that single-stranded DNA is a plasmid replication intermediate.     

Can nuclear localization signals enhance nuclear localization of plasmid DNA?

Nonviral vectors are safer and more cost-effective than viral vectors but are significantly less efficient, and thus, increasing the efficiency of nonviral vectors remains an important objective. One way to overcome this problem is by stimulating the nuclear localization of exogenous genes. Nuclear localization signals (NLSs) are known to be involved in the active transport of exogenous proteins and probes into the nucleus. However, stimulation of nuclear localization of plasmid DNA has yet to be confirmed completely. In the present study, we prepared plasmid DNA-NLS peptide conjugates and adjusted spacer length and number introduced in an attempt to increase transfection efficiency. In comparison to conjugates with unmodified plasmid DNA and short spacers, we found that NLS-plasmid DNA conjugates with covalent bonding by diazo coupling through PEG chain (MW 3400) stimulated complexation with the nuclear transport proteins importin alpha and importin beta. Evaluation of transfection showed higher expression efficiency with plasmid DNA-NLS peptide conjugates than with unmodified plasmids. However, evaluation of intracellular trafficking after microinjection into the cytoplasm showed plasmid DNA-NLS peptide conjugates only within the cytoplasm; there was no NLS-plasmid stimulation of nuclear localization. Our findings suggest that stimulation of plasmid nuclear localization cannot be achieved merely by changing spacer length or chemically modifying plasmid DNA-NLS peptide conjugates. An additional mechanism must be involved.     

Clonal lethality caused by the yeast plasmid 2μ DNA

Strains of Saccharomyces that carry the nib allele of a nuclear gene exhibit a “nibbled” colony morphology if they also harbor the plasmid 2μ DNA. I have found that the expression of the nibbled phenotype is correlated with the presence of a subpopulation of abnormally large cells that give rise to mortal clones. Large cells apparently become large as a consequence of a defect in DNA replication or nuclear division. Large nib cells contain twice as much 2μ DNA per microgram of total DNA as small nib cells do, and elevated 2μ DNA copy number is the cause, not the effect, of increased cell size. It appears that the NIB allele can prevent an increase in 2μ DNA copy number, but cannot produce a decrease once the copy number has exceeded the normal level. I propose, therefore, that the NIB gene product normally represses the amplification of 2μ DNA copy number, and that the nib allele is partially defective in this function.     

Stability during fermentation of a recombinant α-amylase plasmid in Bacillus subtilis

Summary We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus -amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec ⁺ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable.     

YIpDCE1 – an integrating plasmid for dual constitutive expression in yeast

YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerate kinase promoters. YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains. The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens. Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g. modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components). YIpDCE1 has been used to create strains simultaneously overexpressing the permease (ftr1) and oxidase (fet3) components of the yeast high-affinity iron uptake system. This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms.     

Enhanced stability of a 2μ-based recombinant plasmid in diploid yeast

Summary The stability of a 2-based recombinant plasmid, pJDB219, has been compared in glucose-limited chemostat cultures of two haploid strains ofSaccharomyces cerevisiae and a diploid derived from them. The stability of the recombinant plasmid differed in the two haploid hosts but was greatest in the diploid. Enhanced stability in the diploid is probably a function of both the increased copy number and reduced selective burden of the plasmid.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Replication and recombination functions associated with the yeast plasmid, 2μ circle

By examining both the transformation efficiency of yeast of various plasmids containing defined regions of the 2μ circle genome and the characteristics of the resultant transformants, we have identified several regions of the 2μ circle genome which are involved in 2μ circle replication or recombination. First, by identifying those DNA fragments from the molecule which promote high frequency transformation of yeast, we have localized the origin of replication to a sequence partially within the large unique region, which, as determined by subsequent deletion analysis, extends from the middle of the inverted repeat region into the contiguous unique region. Second, by examining the relative efficiency of replication in yeast of hybrid plasmids containing either the entire 2μ circle genome or a fragment of 2μ circle encompassing the origin of replication, we have determined that efficient use of the 2μ circle origin requires some function or functions encoded in the molecule at a site away from the origin. Third, by examining the ability of a mutant 2μ circle molecule to undergo intramolecular recombination in yeast, we have identified a 2μ circle gene which codes for a product required for this process.     

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

BACKGROUND: To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). METHODS AND RESULTS: Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. CONCLUSIONS: Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.     

High concentration culture of Bacillus subtilis spo− mutant strain carrying a recombinant plasmid

A Bacillus subtilis spo⁻ mutant strain harboring a recombinant plasmid was cultured at a high cell density concentration by using a cross-flow filtration system. The cell concentration obtained was 300 as an optical density at 570 nm, which was six times as high as that of a fed-batch culture without using the cross-flow filtration system. However, the specific activity of plasmid-encoded β-galactosidase decreased by filtration. To improve the specific activity, the carbon source was stepwisely changed from glucose to starch. By using a fed-batch culture combined with cross-flow filtration and the stepped change of carbon source, the production of the plasmid-encoded enzyme was improved 3 times compared with that of the fed-batch culture.     

Stability of the recombinant plasmid carrying theBacillus amyloliquefaciens α-amylase gene inB. subtilis

Summary Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110) was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations.     

Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.