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PubMed1968

Nutritional factors influencing the development of competence in the Bacillus subtilis transformation system.

Cultures of Bacillus subtilis developed competence for the uptake of deoxyribonucleic acid in a chemically defined medium with a predictable, reproducible pattern. The gross effects of individual amino acids were determined. Seven amino acids, most of which are reported to be major components of the cell wall, were shown to impair the development of maximal levels of competence. When the synthetic growth medium was supplemented with a mixture of the nine amino acids which we found to stimulate the development of competence, the level of transfection was increased to 10 to 15% of the population. The actual level of competence in these populations was assayed by transformation of unlinked bacterial markers and by two different transfection assays. The results indicate that calculations from cotransfer of unlinked markers overestimates the degree of competence in highly competent populations of B. subtilis, whereas the number of plaques obtained in transfection is an under-estimate of the actual level of competence. The results are interpreted to indicate that neither method of analysis gives a true estimate of the competent population, but that more than 80% of the cells may be competent.

Alanine↗
PubMed2004

Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism.

Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 x 10(5) transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10(-6) to 19 x 10(-6) transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent.

Culture Media↗
PubMed1989

Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12.

The fpg+ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg+ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1::Knr mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Knr mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Knr mutation; (iii) phage P1 mediated transduction of the fpg-1::Knr mutation in the AB1157 background. The resulting fpg- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N'-nitro-N-nitroso-guanidine, (iii) ultraviolet light, (iv) gamma-radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.

Alkylating Agents↗
PubMed2006

Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization.

Complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library, constructed with BamHI in the pECBAC1 vector, contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-transformation-competent binary vector pCLD04541 and contains 84,864 clones, with an average insert size of 137 kb. The two libraries combined contain 192,000 clones and are equivalent to approximately 8.9 haploid genomes of sunflower (3,000 Mb/1C), and provide a greater than 99% probability of obtaining a clone of interest. The frequencies of BAC and BIBAC clones carrying chloroplast or mitochondrial DNA sequences were estimated to be 2.35 and 0.04%, respectively, and insert-empty clones were less than 0.5%. To facilitate chromosome engineering and anchor the sunflower genetic map to its chromosomes, one to three single- or low-copy RFLP markers from each linkage group of sunflower were used to design pairs of overlapping oligonucleotides (overgos). Thirty-six overgos were designed and pooled as probes to screen a subset (5.1x) of the BAC and BIBAC libraries. Of the 36 overgos, 33 (92%) gave at least one positive clone and 3 (8%) failed to hit any clone. As a result, 195 BAC and BIBAC clones representing 19 linkage groups were identified, including 76 BAC clones and 119 BIBAC clones, further verifying the genome coverage and utility of the libraries. These BAC and BIBAC libraries and linkage group-specific clones provide resources essential for comprehensive research of the sunflower genome.

Chromosome Mapping↗
PubMed1983

Genomic cloning and preliminary characterization of the human thymidine kinase gene.

In this report, we describe the cloning of the human cytoplasmic thymidine kinase (tk-C; EC 2.7.1.21) gene and its preliminary characterization. The tk-C sequences were isolated from a phage genomic library made from DNA of a transfected mouse cell carrying the human tk-C gene. The human transforming sequences were identified by homology with human Alu sequences. Six recombinant phages were isolated and five were competent to transfer human TK-C activity to TK-deficient mouse cells when transferred in pairs. Conclusively, sequences homologous to these clones are present in all human TK+ transformants examined. We estimate the maximal size of the tk-C gene to be 14 kilobase pairs and its minimal size to be between 4 and 5 kilobase pairs. The gene contains many noncoding inserts and numerous Alu sequences.

Cloning, Molecular↗
PubMed2004

Transformation of colonial Escherichia coli on solid media.

We report that colonial Escherichia coli cells on various solid media can develop modest genetic competence. Using an on-filter culture system, we found that E. coli colonies on CaCl2-containing agar were transformed in the presence of plasmid DNA. Interestingly, transformation also occurred on LB-agar, various moist foods and even on H2O-agar. These results suggest that some populations of colonial E. coli in various environments could become transformable regardless of the surrounding Ca2+ concentration.

Agar↗
PubMed2005

Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5.

Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, which causes a highly contagious pleuropneumonia. As an adaptation to the iron-restricted environment of the host, A. pleuropneumoniae possesses iron acquisition pathways mediated by surface receptors that specifically bind transferrin from the host. The receptor is composed of two receptor proteins, transferrin-binding protein A and B (TbpA and B), which are both capable of binding to transferrin. An impairment of iron uptake mechanisms is likely to reduce virulence. For this reason, these two proteins can be useful as a candidate target for A. pleuropneumoniae vaccination. To do this, genes encoding the TbpA and B from a serotype 5 isolate of A. pleuropneumoniae were amplified from genomic DNA template by PCR and cloned into a pRSET prokaryotic expression vector, generating the pRSET-A.pp-TbpA and B. Escherichia coli BL21(DE3)pLysS competent cells were transformed with each construct followed by the induction of protein expression by the addition of IPTG. Bands corresponding to the predicted sizes (110 and 60 kDa) were seen on the SDS-PAGE. Polyclonal antibodies raised against recombinant TbpA and B from mice were reacted with bacterial proteins. This result indicates that the recombinant proteins can induce immunological responses and might be useful as candidate targets for A. pleuropneumoniae vaccination.

Actinobacillus pleuropneumoniae↗
PubMed2002

Transformation of Bacillus subtilis in chocolate milk: evidence for low frequency of establishment of cells transformed under non-selective conditions.

Transformation of naturally competent Bacillus subtilis with plasmid was carried out in chocolate milk without antibiotics. Transformed cells were enumerated during the entire growth phase in chocolate milk. When DNA was added to aliquots of a batch culture after different times of incubation, transformation events were detected at all different growth stages. When DNA was added to a batch culture together with the inoculum, transformed cells were detected at the onset of exponential growth. However, apparently no or only limited growth of these transformed cells was observed. To clarify, whether the limitation of growth was due to suppression by non-transformed cells, different proportions of B. subtilis cells either carrying or not carrying the plasmid were mixed and inoculated into chocolate milk without antibiotic. Our results indicate that suppression appears to be of minor importance. Instead, plasmid-bearing cells appear to suffer from a prolonged lag-phase. However, the failure to exhibit significant growth of cells which had taken up the plasmid in chocolate milk appears to be due to failure of these cells to establish themselves as permanently transformed under non-selective conditions.

Animals↗
PubMed1975

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity.

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.

Adenosine Triphosphate↗
PubMed1991

A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product.

Tumour-suppressor genes, such as the human retinoblastoma susceptibility gene (Rb), are widely recognized as being vital in the control of cell growth and tumour formation. This role is indicated, in part, by the suppression of tumorigenicity of human tumour cells after retrovirus-mediated Rb replacement. How Rb acts to bring about this suppression is not clear but one clue is that the Rb protein forms complexes with the transforming oncoproteins of several DNA tumour viruses, and that two regions of Rb essential for such binding frequently contain mutations in tumour cells. These observations suggest that endogenous cellular proteins might exist that bind to the same regions of Rb and thereby mediate its function. We report here the identification of one such human cellular Rb-associated protein of relative molecular mass 46,000 (46K) (RbAP46). Two lines of evidence support the notion that RbAP46 and simian virus 40 T antigen have homologous Rb-binding properties: first, several mutated Rb proteins that failed to bind to T also did not associate with RbAP46; and second, both T antigen and T peptide (amino acids 101-118) were able to compete with RbAP46 for binding to Rb. The apparent targeting of the RbAP46-Rb interaction by oncoproteins of DNA tumour viruses strongly suggests that formation of this complex is functionally important.

Amino Acid Sequence↗
PubMed1996

Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus.

Adenovirus E1 Proteins↗
PubMed1971

Effects of pH on transformation of Bacillus subtilis with single-stranded deoxyribonucleic acid.

Variation in frequencies of transformation mediated by native and single-stranded DNA and its dependence on pH of the medium were investigated. The results indicate that the biological activity of deoxyribonucleic acid (DNA) of both configurations assayed in the presence of ethylenediaminetetraacetic acid (EDTA) increases as the pH of the transformation mixture is lowered from 7.7 to a maximum transformation frequency near pH 6.1. At this lower pH, native DNA transforms equally in medium with and without EDTA, and single-stranded DNA is 0.4 to 0.6 as active as native DNA in transforming Bacillus subtilis. A high efficiency of transformation with single-stranded DNA was observed for five markers in three recipient strains. The increased efficiency of native DNA appears to be caused by a lesser capacity of EDTA to bind magnesium at the lower pH. The increased efficiency of single-stranded DNA at pH below 7.0 results from decreased activity of a single-strand specific nuclease present in competent populations.

Adsorption↗
PubMed1975

Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells.

The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor exogenous DNA upon Ca2+ treatment. At saturating DNA concentrations the cells were capable to take up irreversibly about 6-10(8) dalton of donor DNA per cell (if to assume that all the cells are competent). About 75% of this DNA was found to be attached to the cytoplasmic membrane. Employing Poisson approximation for distribution of the donor-marker DNA molecules on recipient cells it was found that the efficiency of a marker recombination and expression is about 2-10(-5). The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor 0exogenous

Calcium↗