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PubMed1992

Improved method for electroporation of Staphylococcus aureus.

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.

Cell Membrane Permeability↗
PubMed2006

Genomic distribution and functions of uptake signal sequences in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is naturally competent for transformation, with a transformation system similar to that of Haemophilus influenzae that preferentially takes up DNA bearing uptake signal sequences (USS) with the same 9-base USS core. This study examined the function of the extended 29-base USS, which comprises a highly conserved 1st region (containing the 9-base core) and 2nd and 3rd semi-conserved AT-rich regions, in transformation of A. actinomycetemcomitans. Transformation frequency was not affected by either location (in middle or at 5' end) or quantity (one or two) of USS in donor DNA. Relative transformation efficiencies (in comparison to the positive control) were 28-67 % for linear DNA with single-base mutations in the USS 1st region, and 47 % and 73 %, respectively, for linear DNA with USS that contained either a non-consensus 2nd or a non-consensus 3rd region. Plasmids with a stand-alone 1st or a stand-alone 2nd-3rd region exhibited 21 % and 6 % relative transformation efficiencies, respectively. It was also noted that A. actinomycetemcomitans and H. influenzae were similar in the frequencies and distribution patterns of USS in their genomes. In conclusion, all three regions of the extended 29-base USS are required for optimum transformation in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed2002

Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation.

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.

Agrobacterium tumefaciens↗
PubMed2006

Microencapsulated nerve growth factor-expressing NIH3T3 cells-incorporated tissue engineering skin: a preliminary study.

INTRODUCTION: In order to find a suitable carrier to deliver the product of gene transfection to improve the performance of bioengineered dermis, we used microencapsulation and gene transfection technology together for the first time and found that it was feasible. METHODS: We used a recombinant nerve growth factor (pcDNA3.1+/NGF) to modify NIH3T3 cells genetically. Control of NIH3T3-NGF cells were encapsulated within microspheres composed of alginate-poly-L-lysine-alginate and cultivated in-vitro. The concentration of NGF released from the microencapsulated NIH3T3-NGF cells was confirmed using ELISA assay. We co-cultivated microencapsulated NIH3T3-NGF cells, NIH3T3 cells (control) with human keratinocytes and fibroblasts, and tested the percentage of cycle of these cells. The alkaline hydrolysis method was used to analyse the content of hydroxyproline (Hyp). Immunohistochemistry method was used to calculate the transformation efficiency from fibroblasts to myofibroblasts. RESULTS: The concentration of NGF released from the microencapsulated NIH3T3-NGF cells lasted about six weeks in the supernatant of bioengineered dermis in-vitro. The proliferation of keratinocytes, as well as the concentration of Hyp in supernatant of fibroblasts, were promoted about three times. Transformation efficiency from fibroblasts to myofibroblasts was increased approximately two-fold because of the bio-effects of NGF. Two kinds of microencapsulations were seeded into collagen which contained human fibroblasts to form bioengineered skin. Microencapsulated NIH3T3-NGF cells formed a thicker dermis. The concentration of Hyp in the bioengineered skin which indicated the level of collagen synthesis was increased due to existing NGF. CONCLUSION: Microencapsulated NIH3T3-NGF cells can be used to enhance performance of bioengineered dermis and it also can be deduced that other cytokines can be used to treat local wound areas.

Alginates↗
PubMed1988

[Effects of malnutrition in utero and during lactation on various parameters of the small intestine in rats].

Short and long term effects of malnutrition on the small intestine, applied to the rat in uterus and lactation, have been studied. Malnutrition was induced by feeding the pregnant rats on 14 g daily during pregnancy and 21 g during lactation. In the pups (0, 15, 30, 90 and 150 days old), body weight and wet and dry weight and length of small intestine were measured. At 2.5-3 months of age, food transformation efficiency was studied, at 3 and 5 months of age in vivo intestinal absorption of D-glucose (11 mM) was measured. The results indicate a significant decrease in intestinal morphometric parameters in malnourished animals from birth to the age of 5 months. At the age of 3 months both food transformation efficiency and in vivo absorption of glucose were significantly higher in early undernourished animals, whereas at 5 months, glucose absorption was significantly higher in control. It can thus be concluded that early malnutrition altered the small intestine development and functionality and that total recovery did not occur after 4 months on a normal diet.

Animal Nutritional Physiological Phenomena↗
PubMed2003

Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection.

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

Biolistics↗
PubMed1982

DNA-mediated gene transfer in Chinese hamster ovary cells: clonal variation in transfer efficiency.

Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies greater than or equal to 3 X 10(-4) were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk+ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.

Animals↗
PubMed2002

Difference between deoxyribose- and tetrahydrofuran-type abasic sites in the in vivo mutagenic responses in yeast.

We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay. Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorporation opposite abasic sites was analyzed. Cytosine was most frequently incorporated opposite a natural abasic site (O) ('C-rule'), followed by thymine. Deletion of REV1 decreased the transformation efficiency and the incorporation of cytosine nearly to a background level. In contrast, deletion of RAD30 did not affect them. We compared the mutagenic specificity with that of a tetrahydrofuran abasic site (F), an abasic analog used widely. Its mutation spectrum was clearly different from that of O. Adenine, not cytosine, was most favorably incorporated. However, deletion of REV1 decreased the transformation efficiency with F-containing oligonucleotide as in the case of O. These results suggest that the bypass mechanism of F is different from that of O, although the bypasses in both cases are dependent on REV1. We also found that the mutagenic specificity of F can be affected by not only the adjacent bases, but also a base located two positions away from F.

DNA↗
PubMed2003

Latent membrane protein 1 is critical for efficient growth transformation of human B cells by epstein-barr virus.

The EBV latent membrane protein 1 (LMP1) is an integral membrane protein that acts like a constitutively activated receptor. LMP1 interacts with members of the tumor necrosis factor receptor-associated factor family, as well as with tumor necrosis factor receptor-associated death domain, resulting in induction of nuclear factor-kappaB, the p38 mitogen-activated protein kinase pathway, and the c-Jun NH(2)-terminal kinase activator protein 1-signaling cascade. The binding of Janus kinase 3 results in activation of signal transducers and activators of transcription. The domain structure of LMP1 has been mapped extensively, but the quantitative contribution of distinct LMP1 domains to the efficiency of B-cell proliferation by EBV has not been determined. On the basis of the maxi-EBV system, which allows us to introduce and study mutations in the context of the complete EBV genome, a panel of 10 EBV mutants with alterations in the LMP1 gene locus was established. The mutant EBVs were tested for their efficiency to induce and maintain proliferation of clonal B-cell lines in vitro. Surprisingly and with reduced frequency, EBV mutants which deleted LMP1's COOH terminus, transmembrane domains, or the entire open reading frame were able to generate proliferating B-cell clones that were dependent on the presence of human fibroblast feeder cells. A B-cell clone carrying the LMP1-null mutant EBV genome was also analyzed for oncogenicity in severe combined immunodeficiency mice. Our results demonstrate that LMP1 is critical but not mandatory for the generation of proliferating B cells in vitro. LMP1 functions greatly contribute to EBV's transformation potential and appear essential for its oncogenicity in severe combined immunodeficiency mice.

Alleles↗
PubMed1986

Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.

Animals↗
PubMed1984

Binary Agrobacterium vectors for plant transformation.

A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.

Amino Acid Oxidoreductases↗
PubMed1994

Isolation and characterization of deletion derivatives of pDL282, an Actinobacillus actinomycetemcomitans/Escherichia coli shuttle plasmid.

Recent reports have described the construction of several shuttle plasmids (D.J. LeBlanc, L. L. Lee, A. Al-Jaibat, P. K. Sreenivasan, and P. M. Fives-Taylor, Oral Micro. Immunol. (1993) 8, 94-99) and the development of an efficient transformation system for Actinobacillus actinomycetemcomitans (P. K. Sreenivasan, D. J. LeBlanc, L. L. Lee, and P. M. Fives-Taylor, Infect. Immun. (1991) 59, 4621-4627), a gram-negative bacterium implicated in human periodontal disease. This report presents results from further studies on pDL282, an A. actinomycetemcomitans-Escherichia coli shuttle plasmid. A. actinomycetemcomitans containing pDL282 lost the plasmid at a rapid rate when cultured in antibiotic free medium. Intact pDL282 was maintained for 20 generations or more when the host cells were grown in the presence of ampicillin, or ampicillin plus spectinomycin. However, prolonged incubation in the presence of spectinomycin only resulted in the emergence of one or the other of two unique deletion derivatives of pDL282, designated pPK1 and pPK2. Whereas A. actinomycetemcomitans was efficiently transformed with pDL282 (5.7 kb), pPK1 (3.6 kb), and pPK2 (2.5 kb), E. coli was transformed only by the two largest species. Like the parent molecule, pPK1 and pPK2 were rapidly lost from A. actinomycetemcomitans hosts in the absence of antibiotic selection. Neither pPK1 nor pPK2 suffered any further deletions following prolonged cultivation in the presence of spectinomycin. The minimal replicon of pVT736-1, the A. actinomycetemcomitans-derived plasmid component of pDL282, was located within a 1400 bp DNA fragment of pPK1 and pPK2.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus↗
PubMed1963

OPTIMAL CONDITIONS FOR THE TRANSFORMATION OF STREPTOCOCCI.

Perry, Dennis (Northwestern University Medical School, Chicago, Ill.) and Hutton D. Slade. Optimal conditions for the transformation of streptococci. J. Bacteriol. 85:636-642. 1963.-A study of the properties of the streptococcal transforming system, employing streptomycin resistance as a marker, resulted in a 1,000- to 10,000-fold increase in the rate of transformation. In some cases, as high as 0.5% transformants have been obtained. Certain aspects of the system differed markedly from those of other bacterial transforming systems, particularly with regard to the time for the appearance of competence. When a group H strain was exposed to deoxyribonucleic acid after various periods of incubation, the time at which cells were most competent was about 2 hr. Similar experiments with a different group H strain and an ungroupable strain showed that competence for both occurred after 1 hr of growth. It is significant that the period of optimal competence, though attained at different times, took place at the beginning of the logarithmic phase of growth. The temperature optimum for transformation was about 37 C. Cells preincubated in sheepblood broth exhibited a higher level of transformation than cells preincubated in plain or human serum broth. That some correlation exists between the attainment of competence and growth is evident. In contrast, human serum was most effective in the transforming media. The few preparations of bovine albumin (fraction V) employed did not appreciably support transformation. Data are also presented which show that some relationship exists between transforming efficiency and serological classification, in that homologous transformation showed a 100- to 10,000-fold greater efficiency over heterologous transformation. Antibodies to deoxyribonuclease, as well as other inhibitors of deoxyribonuclease, failed to bring about transformation in group A streptococci.

Animals↗
PubMed2005

A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase.

The present paper reports a highly efficient method of making blunt ends from cohesive ends of double-stranded DNA. Klenow fragment and Pfu DNA polymerases were used to fill in the cohesive ends. Since the transformation efficiency can directly reflect the filling-in efficiency, similar ligation and transformation conditions were used, and the filling-in efficiency was compared with the corresponding transformation efficiency. The results indicate that the filling-in efficiency of Pfu DNA polymerase was 1.96 times that of Klenow fragment and its efficiency was markedly higher than that of Klenow fragment (P<0.01). The optimization experiments on reaction conditions indicate, when the pH is 8.5 and the temperature is 74 degrees C, that the filling-in efficiency was highest upon using a buffer containing 3 mM MgSO4 and 300 microM dNTP.

Buffers↗
PubMed2006

Agrobacterium-mediated delivery of marker genes to Phanerochaete chrysosporium mycelial pellets: a model transformation system for white-rot fungi.

The lack of an efficient transformation system in filamentous fungi is one of the major problems in carrying out cloning and expression of genes to produce a value-added product at the commercial level. Therefore an attempt has been made to develop an efficient, convenient and expeditious genetic transformation system for successful transfer of transferred DNA, carrying the genes coding for GUS (beta-glucuronidase) (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of Phanerochaete chrysosporium, a widely studied lignin-degrading white-rot fungus. The transformation of the marker gene was confirmed through GUS, PCR and Southern-blot hybridization. Transformation with acetosyringone, in the presence of light with partial bleaching for 1 h, were found to be a better combination for fungal transformation with 48% efficiency. The agro-transformation method to transform the modified mycelial pellets offers a practical means for exploiting transgenic approaches in genetic manipulation and improvement of P. chrysosporium, a model white-rot fungus. Moreover, it has the potential to overcome the technical hurdles in genetic manipulation to bio-technologically exploit many other filamentous fungi of industrial importance.

Gene Targeting↗
PubMed2002

Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection.

Here we report on the development of a new dominant selection marker for plastid transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved via homologous recombination, and plastid transformants were recovered on the basis of their resistance to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the 5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile and showed maternal inheritance of the transplastome in the progeny.

Acinetobacter baumannii↗
PubMed1999

Low-level transforming activity of an activated Ras gene under the control of a vaccinia virus p40 promoter is abrogated by truncation of the Ras cDNA.

Many human cancers have been shown to contain activated forms of the Ras proto-oncogene. Mutations comprising amino acid changes at codons 12, 13 and 61 therefore represent unique targets for cancer immunotherapy. Recombinant Vaccinia viruses encoding point mutated Ras oncogenes have raised issues concerning the safety and transforming ability of these recombinant vaccines. Vaccinia virus, a representative of the orthopox virus genus, is a large DNA virus that is cytopathogenic and that replicates in the cytoplasm of the infected cell. However, it remains unclear whether orthopox viruses are capable of genetic interactions with infected cells. Our studies show that DNA isolated from cells infected with a recombinant Vaccinia virus expressing mutated Ras constituted a poor reagent for transfection into NIH3T3 cells for transformation analysis. Stable integration of a recombinant Vaccinia virus expressing mutant Ras DNA was not detected in recipient cells. This study also demonstrates that the crossover plasmids used to generate the recombinant virus where the activated Ras gene is under the control of a Vaccinia virus early promoter had low but detectable transforming efficiency in the NIH3T3 transformation assay. Analysis of the transfected cells indicated that Ras transcription was initiated upstream of the Vaccinia virus promoter. The introduction of wobble mutations as well as the truncation of the Ras protein removed the transforming capabilities of the crossover vector. This study demonstrates the potential problems and solutions in the use of point mutated oncogenes in live vectors for cancer vaccine development.

3T3 Cells↗