Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Transformation Competence”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources
PubMed1995

Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.

Amino Acid Sequence↗
PubMed1989

Assembly of genes from partially overlapping fragments using single-stranded DNA and sequence-specific synthetic oligodeoxynucleotides.

A simple procedure for the precise assembly of functional DNA sequences from overlapping fragments is described. The fragments to be joined are cloned in tandem in the proper relative orientation into a vector from which single-stranded DNA copies can be obtained. Single-stranded DNA is cut by a restriction enzyme at corresponding sites in the two overlap regions, which are made double-stranded by annealing an oligonucleotide of appropriate sequence to them. This results in the excision of the unwanted sequences between the two overlap regions. After removal of the restriction enzyme the DNA is reannealed using the same oligonucleotide, ligated to give closed circular molecules and used to transform competent cells. Clones with the desired structure appear in the progeny at high frequency. The method has the advantage that restriction enzymes with short recognition sequences, cutting frequently in the target DNA, can be used and hence the overlap region required can be quite short.

Base Sequence↗
PubMed1980

Switches in macromolecular synthesis during induction of competence for transformation of Streptococcus sanguis.

The induction of synchronous development of competence for genetic transformation in Streptococcus sanguis, by either endogenous or exogenous competence factor (CF), is manifested in the transient synthesis of a new set of at least 10 polypeptides, ranging from 14,000 to 51,000 in molecular weight. Eight polypeptides (E14, E16, E24, E28, E32, E37, E44, E51) appear early, and two polypeptides (L34, L42) appear 5-10 min later. One of the newly synthesized early polypeptides, E16, is shown to be a component of the presynaptic complex containing single-stranded DNA that is produced in vivo upon uptake of native donor DNA. Concomitant with this induced synthesis of competence-specific polypeptides there is a net decrease in RNA and protein synthesis but no change in DNA synthesis; donor DNA-binding ability and transformability reach maxima during the phase of diminishing macromolecular synthesis. Subsequently, donor DNA-binding ability and transformability decay at disproportionate rates as cells return to the normal state of macromolecular synthesis within one generation. Coincident with the induction of competence, the synthesis of a new RNA transcript of high molecular weight appears to be induced which continues during the restricted phase of total cellular RNA synthesis.

Bacterial Proteins↗
PubMed2003

The genome of herpesvirus saimiri C488 which is capable of transforming human T cells.

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.

Amino Acid Sequence↗
PubMed1983

Taxonomic implications of quantitative transformation in Acinetobacter calcoaceticus.

Quantitative transformation of streptomycin resistance marker was carried out with strains of Acinetobacter calcoaceticus. Standard recipient strain was the competent BD4-Ss. Transformation proceeded in a liquid system with a concentration of 20 micrograms/ml of streptomycin resistant DNA (Sr-DNA) from BD4, 17 reference strains of Acinetobacter, and 42 recent clinical isolates of the acid forming variant (anitratus) and 12 of the non-acid forming variant (lwoffi) as donors of Sr-DNA. The exposure time was 20 min before interruption with DNase and quantitated plating. Among the strains examined were differentiated 16 biotypes as based on oxidative acid production from glucose and lactose, haemolysis, urease, gelatinase, and growth with citrate as the sole carbon source. Autologous transformation gave a transformation of 2.94 (+/- 0.66)% of the recipient cells (quantitated by colony forming units). Sr-DNA from 50 of 63 strains were able to transform BD4-Ss. The transformation ratio was 0.06-6.4% of autologous BD4 transformation. Two further recent clinical isolates were weakly transformation competent. Competence was only found in autologous transformation. The major portion (50/63) of the strains belong to the BD4 genotype of A. calcoaceticus, but the taxonomic position of the 13 non-donors in respect to BD4 could not be evaluated by transformation because of lack of a competent recipient. The strains transforming BD4 belonged to both the glucose acidifyers and the non-acidifyers without genetic distinction. The results are consistent with recognition of both as belonging to the same species, but with their recognition as species variants: A calcoaceticus var, anitratum and A. calcoaceticus var. lwoffi.

Acinetobacter↗
PubMed2006

Functional characterization of the competence protein DprA/Smf in Escherichia coli.

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.

Bacterial Proteins↗
PubMed1984

Cloning of the rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned DNA sequence in normal rat kidney cells by microinjection.

By the use of recombinant DNA technology and microinjection in cultured cells, the molecular genetic elements involved in the evolution of a retrovirus with the multipotential to infect, transform and replicate in host cell, have been critically examined in this investigation. Recently we have identified and purified the integrated and proviral DNA sequences specific for two rat endogenous helper leukemia viruses, WR- RaLV , originated from a chemically induced wild rat fibrosarcoma, and RHHV , isolated from a chemically induced rat hepatoma, HTC-H1 (1). By using a multidisciplinary approach combining restriction endonuclease analysis, reverse phase V-column chromatography, agarose gel electrophoresis, Southern blot transfer and filter nucleic acid hybridization, we were able to demonstrate that the rat helper leukemia viral DNA sequence was approximately 8.4-8.8 kb. The 8.8 kb RHHV DNA was molecularly cloned via the EK-1 certified vector pBR 322 plasmid into E. coli RRI cells. A successful recombinant clone, 8/32, that carried one entire RHHV 8.8 kb DNA sequence was mapped by restriction endonuclease analyses. Restricted DNA fragments of various sizes throughout the complete RHHV genome were isolated and purified for intranuclear microinjection into normal rat kidney cells. Release of type C infectious helper virus in these microinjected cells was investigated by superinfection on K-NRK, Kirsten sarcoma transformed non-producer cells. Recombination of the helper viral DNA sequence, en toto or of subgenomic sizes, carried in microinjected cells, with the sarcomagenic DNA sequence, carried in K-NRK cells, was also studied by genome-rescue and cell-transformation experiments. Our observations led to the conclusion that all critical genetic elements including the 5' LTR helper DNA sequence, gag, pol, and env genes, encoded for the biological activity of the type C helper virus resided within the 6.0 kb proximal to the 5' terminus of the endogenous rat type C helper virus DNA. They proved vitally essential for the recombination with the Src sequence during the evolution of an infectious, transforming and replication-competent retrovirus.

Animals↗
PubMed1995

Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA.

To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.

Amino Acid Sequence↗
PubMed2003

[Highly efficient construction of recombinant adenovirus containing double suicide gene driven by cytomegalovirus promoter using two-step CaCl2 transformation method].

OBJECTIVE: To construct recombinant adenovirus containing double suicide gene driven by cytomegalovirus (CMV) promoter. METHODS: CD and TK gene were amplified by PCR and cloned into the shuttle vector pAdtrack CMV, and the resultant plasmid pAdtrack CMV-CDTK was linearized with PmeI and transformed into competent AdEasier-1 cells prepared by CaCl2 method. PacI digestion of identified recombinant plasmid DNA was performed before it was transected into 293 cells to package adenovirus, which was used to infect endothelial cells in vitro after adenovirus titer determination. RESULTS: Chemical transformation of linearized transfer plasmid into AdEasier-1 cells resulted in very high ratio (20/20) of positive clones of recombinant adenovirus containing. CD and TK fusion gene as confirmed by PCR test. CONCLUSION: The modified AdEasy system is more convenient and efficient for constructing recombinant adenovirus, which may facilitate further study of anti-tumor therapy targeting at the double suicide gene.

Adenoviridae↗
PubMed1981

Mutagenesis during transformation of Bacillus subtilis. I. An increase in "selfing' resulting from hybrid donor DNAs.

Reverse mutations increase when competent Bacillus subtilis cells are transformed with high concentrations of homologous "selfer' DNA. A high proportion of the mutants were also transformants of linked genes. A stimulation in the appearance of reversed mutations occurred when homoduplex and heteroduplex "selfer' DNAs were used as donors. Digestions of native and hybrid DNAs with nuclease S1 from Aspergillus oryzae resulted in the preferential decrease of mutations as compared to a much smaller inactivation of single marker transformation. Among various repair-deficient strains of B. subtilis, only poly A mutants showed a preferential effect of either suppressing or stimulating the frequency of reverse mutation induced by "selfer' DNA. The results are consistent with mutagenic errors occurring during gap-filling steps in the process of either mismatch repair or recombinational strand exchanges.

Aspergillus oryzae↗
PubMed1982

Marker rescue by a homologous recipient plasmid during transformation of gonococci by a hybrid Pcr plasmid.

A 42-kilobase hybrid Pcr plasmid (pFA14) was formed when the naturally occurring 7.2-kilobase Pcr plasmid pFA3 was introduced by transformation into a competent gonococcal recipient containing the 36-kilobase conjugative plasmid pFA2 (Sox et al., J. Bacteriol. 138:510-518). Analysis of the structure of pFA14 showed that it was a stable recombinant between pFA3 and pFA2. The transformation efficiency of pFA14 was increased 300- to 10,000-fold by the presence in isogenic recipients of the homologous plasmid pFA2. The presence of a homologous plasmid in the recipient also markedly increased the likelihood of recovery of intact donor-size Pcr plasmids in the transformants. The presence of pFA2 had no effect on the competence of piliated or nonpiliated gonococci for transformation by either linear chromosomal DNA or a nonhomologous Pcr plasmid. Increased transformation efficiency of the hybrid Pcr plasmid pFA14 may have been due to recombination between the nicked or linearized donor plasmid and the homologous recipient plasmid (marker rescue).

Chromosomes, Bacterial↗
PubMedDate not supplied

[The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli].

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.

DNA, Bacterial↗
PubMed1998

Construction of a quantitative PCR system to determine expression of tumor associated antigen.

Cancer immunotherapy is a strategy for cancer treatment by induction of anti tumor responses. The identification of candidate tumor associated antigens (TAA) suggested their potential use as immunogens for vaccination studies. Quantification of a TAA expression by cancerous cells is an important factor in determination of induced immune response efficiency against tumors and thus enables us to devise optimal immunotherapy protocol to cure cancer. The quantitative polymerase chain reaction (PCR) enables us to compare the TAA expression in highly metastatic tumor clones with that in less metastatic ones and in normal cells. It allows us to gain more insight into genome rearrangements that occur in malignant transformation as well as broaden our knowledge about tumor cell gene expression regulation. One of the peptides isolated previously in our lab from a murine lung carcinoma is the mutated Connexin 37 (cx37), a gap-junction protein. Research is underway to determine the expression level of the TAA in various Lewis lung carcinoma cells. This evaluation is achieved by means of quantitative PCR. A quantitative PCR experiment includes preparation of a control template, which is added in known amounts together with the target template in a series of amplification reactions. The control template uses the same primers as the target sequence, yet their PCR products differ in size so as to be distinguishable. Two methods were used to produce this control template. The first one included specific deletion of a sequence of approximately 100 bp that lay between the two primers, insertion of the new template into a plasmid vector, transformation of competent bacteria, detection of transformed bacterial colonies and isolation of the plasmid DNA in a large quantity. The non-mutated, deleted Connexin 37 cDNA was also isolated from bacteria and used for another experiment aimed at producing deleted, mutated Connexin 37 cDNA by means of primer mutagenesis. (Fig. 5, Ref 8.)

Animals↗
PubMed1970

Nature of the ethylenediaminetetraacetic acid requirement for transformation of Bacillus subtilis with single-stranded deoxyribonucleic acid.

The ethylenediaminetetraacetate (EDTA) requirement for transformation of Bacillus subtilis with single-stranded deoxyribonucleic acid (DNA) was examined. The results indicate that a chelating agent such as EDTA is a stringent requirement for transformation with single DNA strands only at nonsaturating DNA concentrations, and that EDTA, when required, must be present during several steps in the transformation process and appears to insure the survival of single-stranded DNA by rendering a nuclease in competent populations inactive.

Bacillus subtilis↗
PubMed1991

Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.

Amino Acid Sequence↗
PubMed1976

Discrimination of competent Bacillus subtilis with respect to ribonucleic acids.

A study has been made of the affinity of double-stranded helical RNA for DNA receptors in competent Bacillus subtilis. In competition experiments, using homologous and heterologous DNA samples which had been sheared to molecular weights comparable to that of the RNA (about 2 x 10(6)), and which still exhibited appreciable competition in DNA uptake experiments, the replicative form of phage f2 RNA showed no evidence of affinity for receptor sites. A second double-stranded RNA preperation from a widely different source, a mycophage of Penicillium chrysogenum, behaved similarly to the f2 RNA. Transfer RNA and 23S ribosomal RNA, which reduce transformation frequencies in pneumococcus, did not compete for B. subtilis receptors. Lack of competition was not due to enzymatic degradation of the RNA, since the latter was recovered intact following exposure to competent cells. Under conditions where homologous native DNA undergoes normal uptake, there was virtually no uptake of native double-stranded RNA. The results are examined in the light of reports on transformation by RNA and DNA-RNA hybrids, and also in relation to the characterization of the specificity of cell-nucleic acid interactions.

Bacillus subtilis↗
PubMed1973

Transformation and transfection in lysogenic strains of Bacillus subtilis 168.

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.

Bacillus subtilis↗
PubMed1995

Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916.

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of "corncobs", a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.

Adhesins, Bacterial↗