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PubMed1972

Competence mutants. II. Physical and biological fate of donor transforming deoxyribonucleic acid.

Transformation-deficient (com(-)) mutants, which are able to bind donor transforming deoxyribonucleic acid (DNA) without yielding a significant number of transformants, were studied with regard to the fate of donor DNA. In no case was there any detectable degradation into acid-soluble radioactivity after donor DNA uptake. Physical experiments showed that some of these mutants are deficient in their ability to associate donor DNA with the recipient's chromosome (dad(-) mutants, for donor association defective), whereas others are able to form what appear to be normal donor-recipient complexes. In spite of physical evidence for integration, none of the dad(-) mutants contains biologically active recombinant DNA, suggesting that they might be deficient in the recombination process (dab(-) mutants, for donor association biologically defective). Donor biological activity is not replicated in any of the mutant strains, and in some cases there is a 10-fold reduction of donor transforming DNA within 60 min after DNA uptake.

Bacteriological Techniques↗
PubMed2001

High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.

Cryopreservation↗
PubMed1995

The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction.

The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation. Increased expression was associated with the appearance of a recA-specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (about 4.3 kb) transcript detected from non-competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA- lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.

Bacterial Proteins↗
PubMed1978

Synchronous division and rates of macromolecular synthesis in Haemophilus influenzae competent for genetic transformation.

When competent Haemophilus influenzae were returned to complete growth medium, they resumed growth at once and underwent a synchronous division after 20 to 25 min. The rate of DNA synthesis increased to the definitive rate very quickly after resumption of growth. In contrast, the rate of RNA synthesis remained low until the time of division, at which time it accelerated dramatically. Changes in the rate of protein synthesis were similar in form to those of RNA synthesis. The kinetics of loss of competence upon resumption of growth had a pattern similar to the rate of RNA synthesis, and both processes accelerated markedly at the time of cell division, suggesting an inverse relationship between RNA synthesis and the ability to take up DNA.

Bacterial Proteins↗
PubMed1990

Induction of replicative competence ("priming") in normal liver.

We have used a system of nutritional manipulation to investigate whether hepatocytes of the normal liver can be primed for replication in vivo. In this system, rats that are denied protein for 3 days undergo a burst of hepatic DNA synthesis and mitosis when they are refed amino acids, while normally fed or starved rats do not respond. To determine if hepatocytes of protein deprived (PD) rats have been "primed" for replication, we examined changes in protooncogene expression in livers of PD rats to see if they would mimic the pattern of gene expression that is induced early after partial hepatectomy. c-jun, c-myc, and p53 mRNAs were elevated in livers of PD rats, while c-fos and c-ras genes were not expressed. The administration of amino acids to PD rats stimulated hepatic DNA synthesis in a shorter period than is required after partial hepatectomy and induced p53 and c-ras expression. In culture, hepatocytes from PD rats had higher levels of c-myc mRNA, underwent morphological changes more rapidly, and reached maximum rates of DNA synthesis earlier than normal hepatocytes. In both normal and primed hepatocyte cultures, transforming growth factor alpha stimulated DNA synthesis more effectively than epidermal growth factor. We conclude that hepatocytes pass through a priming stage before they proliferate and that replicative competence without DNA synthesis can be induced in hepatocytes in the normal liver.

Amino Acids↗
PubMed1999

An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence.

Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 10(5) transformants per microg of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria.

Blotting, Southern↗
PubMed2005

Design and development of an internal control plasmid for the detection of Mycobacterium avium subsp. paratuberculosis using real-time PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants. The hspX gene and insertion sequence IS900 can be used to diagnose Johne's with PCR. Generally, a single PCR tube containing the DNA sequence of interest is run as a positive control with each set of reactions. Single reactions within a PCR run can fail while the positive control does not. Thus, a single positive control tube does not determine if all PCR reactions worked properly. Our objective was to construct a plasmid to use as an internal control in each reaction. A plasmid containing an insert of M. bovis-hspX-M. bovis DNA was modified to remove a portion of the hspX insert used by the reverse hspX primer. The remaining insert was ligated back together and transformed into competent cells. Sequencing confirmed removal of 71 bp. PCR reactions using three primers (TB/M. bovis reverse, hspX forward and reverse) for hspX gene detection and four primers (IS900 forward and reverse, hspX forward, and TB/M. bovis reverse) for IS900 detection were optimized by titrating various amounts of plasmid against varied amounts of MAP genomic DNA. Plasmid insert amplification confirms a successful PCR reaction and identifies true positives and negatives within each individual reaction. The optimal plasmid amounts are 10 fg/reaction (hspX detection) and 1 fg/reaction (IS900 detection).

Animals↗
PubMed2002

Production of phage-displayed anti-idiotypic antibody single chain variable fragments to MG7 monoclonal antibody directed against gastric carcinoma.

OBJECTIVE: To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer. METHODS: Balb/c mice were immunized i.p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. RESULTS: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed beta or gamma type anti-Id ScFv. CONCLUSION: The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Animals↗
PubMed2006

The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation.

The gene slr0388 was previously annotated to encode a hypothetical protein in Synechocystis sp. strain PCC 6803. When a positively phototactic strain of this cyanobacterium was insertionally inactivated at slr0388, the mutants were not transformable, and appeared to aggregate as a result of increased bundling of type IV pili. Also, these mutants were rendered non-phototactic compared to the wild-type. Quantitative real-time PCR revealed a 3.5-fold increase in pilA1 transcript levels in the mutant over wild-type cells, while there were no changes in the level of pilT1 and comA transcripts. Supernatant from mutant liquid culture contained more PilA1 protein, confirmed by mass spectrometric analysis, compared to the wild-type cells, which corresponded to the increase in pilA1 transcripts. The increase in PilA1 subunits may contribute to the bundling morphology of pili that was observed, which in turn may act to retard DNA uptake by hindering the retraction of pili. This gene is therefore proposed to be designated comF, as it possesses a phosphoribosyltransferase domain, a distinguishing feature of other ComF proteins of naturally transformable heterotrophic bacteria. This report is the second of a competence-related gene from Synechocystis sp. strain PCC 6803, the product of which does not show homology to other well-studied type IV pili proteins.

Amino Acid Sequence↗
PubMed1992

Platelet-derived growth factor (PDGF) accelerates induction of competence, and heparin does not inhibit PDGF-induced competence in primary cultured smooth muscle cells of rat aorta.

The time-dependent effect of platelet-derived growth factor (PDGF) on cell proliferation was investigated to clarify whether PDGF accelerates the rate of proliferation or its start in primary cultured smooth muscle cells (SMC) of rat aorta. In synchronized SMC at the G0 phase, 1, 10 and 100 ng/ml PDGF started DNA synthesis at 24, 15-18 and 12 hr, respectively, after stimulation by 3% fetal bovine serum (FBS) or hypophysectomized rat plasma (deficient in insulin-like growth factor-I (IGF-I)). Heparin (1, 10 or 100 micrograms/ml) decreased only the rate of DNA synthesis stimulated by PDGF in synchronized SMC. DNA synthesis in non-synchronized cells stimulated by PDGF with FBS was determined up to 10 days in culture. The stimulation with 1% FBS plus 30 ng/ml PDGF potentiated the DNA synthesis which was saturated with stimulation by 10% FBS alone, suggesting that prolonged treatment of PDGF transforms SMC. These results demonstrated that PDGF concentration-dependently accelerated the induction of competence independently of IGF-I, and heparin did not inhibit PDGF-induced competence but inhibited progression in primary cultured SMC of rat aorta.

Animals↗
PubMed1972

Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor.

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.

Bacillus subtilis↗
PubMed1988

Strategies for the development of bacterial transformation systems.

An effective transformation system is a prerequisite for facile genetic manipulation of bacteria. Bacteria may be naturally competent for transformation or may be treated with various agents, such as Tris buffers or divalent metal ions, to induce competence. Transformation can also be accomplished by electroporation, or by fusion of protoplasts with PEG in the presence of transforming DNA. Unfortunately, the mechanism by which cells become permeable to DNA and the process by which DNA enters the cells is frequently unknown. In order to establish a transformation system for an untransformable bacterium, recipient strains and transforming DNA must be carefully selected. Since it is impossible to predict in advance which method of transformation will be successful with a particular bacterial strain, several techniques are usually evaluated. This review describes a number of factors that appear to be critical for developing a transformation system and presents a strategy for experimentation with novel bacteria.

Bacteria↗
PubMed2001

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Cell Death↗
PubMed2000

Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer.

Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA. However, we demonstrate that there are strong barriers to transformation of H. pylori strains by plasmids derived from unrelated strains. We sought to determine the molecular mechanisms underlying these barriers. Transformation efficiency was assessed using pHP1, an Escherichia coli-H. pylori shuttle vector conferring kanamycin resistance. Transformation of 33 H. pylori strains was attempted with pHP1 purified from either E. coli or H. pylori, and was successfully introduced into only 11 strains. Digestion of H. pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains. The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H. pylori strains. When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H. pylori strains. Parallel studies with pHP1 from dam+ and dam- E. coli strains confirmed these findings. These data indicate that the endogenous REs of H. pylori strains represent a critical barrier to interstrain plasmid transfer among H. pylori.

Chromobox Protein Homolog 5↗
PubMed1984

Transforming functions associated with Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms the human B-lymphocytes it infects into lymphoblasts that are competent to proliferate indefinitely in tissue culture [1]. The dividing transformed lymphoblasts carry multiple and complete copies of EBV DNA as plasmids [1]. No viral functions that are necessary for EBV-induced transformation have been identified and characterized to date. We have developed an assay that aids in the identification of viral functions needed to maintain the viral plasmid in transformed cells. The assay employs a plasmid vector that encodes resistance to ampicillin and can replicate in E. coli. It also encodes resistance to the neomycin derivative G418, so that its presence can be selected in mammalian cells [2]. Fragments of viral DNA that span the genome have been molecularly cloned into this vector. These recombinant molecules have been transfected into four cell types, and survivors to G418 have been scored. The DNA from one region of EBV gives a 10- to 100-fold higher rate of survival per microgram of added DNA than does the DNA from all other recombinants, as tested in cells that already contain EBV genomes. This recombinant fragment of EBV is maintained in an apparently unrearranged state as a plasmid and therefore carries at least those cis-acting viral elements necessary for plasmid replication.

B-Lymphocytes↗