SEARCH · Search PubMed
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.
The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.
Reticuloendotheliosis virus is an avian type C retrovirus that is capable of transforming fibroblasts and hematopoietic cells both in vivo and in vitro. This virus is highly related to the three other members of the reticuloendotheliosis virus group, including spleen necrosis virus, but it is apparently unrelated to the avian leukosis-sarcoma virus family. Previous studies have shown that it consists of a replication-competent helper virus (designated REV-A) and a defective component (designated REV) that is responsible for transformation. In this study we used restriction endonuclease mapping and heteroduplex analysis to characterize the proviral DNAs of REV-A and REV. Both producer and nonproducer transformed chicken spleen cells were used as sources of REV proviral DNA; this genome was mapped in detail, and fragments of it were cloned in lambdagtWES.lambdaB. The infected canine thymus line Cf2Th(REV-A) was used as a source of REV-A proviral DNA. The restriction maps and heteroduplexes of the REV and REV-A genomes showed that (proceeding from 5' to 3') (i) REV contains a large fraction of the REV-A gag gene (assuming a gene order of gag-pol-env and gene sizes similar to those of other type C viruses), for the two genomes are very similar over a distance of 2.1 kilobases beginning at their 5' termini; (ii) most or all of REV-A pol is deleted in REV; (iii) REV contains a 1.1 kilobase segment derived from the 3' end of REV-A pol or the 5' end of env or both; (iv) this env region in REV is followed by a 1.9-kilobase segment which is unrelated to REV-A; and (v) the helper-unrelated segment of REV extends essentially all of the way to the beginning of the 3' long terminal repeat. Therefore, like avian myeloblastosis virus but unlike the other avian acute leukemia viruses and most mammalian and avian sarcoma viruses, REV appears to be an env gene recombinant. We also found that the REV-specific segment is derived from avian DNA, for a cloned REV fragment was able to hybridize with the DNA from an uninfected chicken. Therefore, like the other acute transforming viruses, REV appears to be the product of recombination between a replication-competent virus and host DNA. Two other defective genomes in virus-producing chicken cells were also cloned and characterized. One was very similar to REV in its presumptive gag and env segments, but instead of a host-derived insertion it contained additional env sequences. The second was similar (but not identical) to the first in its gag and env regions and appeared to contain an additional 1-kilobase inversion of REV-A sequences.
Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.
Explore the source record for details and available documents.
Deoxyribonucleic acid (DNA) from 250 strains of aerobic, nonfermentative, gram-negative coccobacilli and rods were tested for the ability to transform a stable competent auxotroph of Acinetobacter (strain trp E 27) to prototrophy by using the method established by Juni. Several modifications of Juni's original procedure were made to adapt it for use in a clinical diagnostic laboratory. These modifications were directed primarily towards shortening the procedure to allow completion in a time framework consistent with current procedures. The modifications included changes in sterilization temperature, incubation time and temperature of the competent auxotroph and DNA preparation, overnight incubation temperature, and variations in the age of the auxotroph culture when used. Under these conditions, the transformation can easily be performed in 24 h, the final 16 to 18 h being an overnight uninterrupted incubation period. When used in conjunction with the glucose oxidative fermentative basal metabolism test, it provided a rapid highly efficient means for grouping and identifying acinetobacters which is far superior to a biochemical schema. Without exception, the 141 strains of DNA from Acinetobacter species were able to transform the auxotroph to prototrophy. None of the 105 oxidase-positive nonfermenters possessed DNA which was able to transform the Acinetobacter auxotroph to prototrophy.
As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.
The genetic variability in comC, the gene encoding the quorum-sensing molecule, competence-stimulating peptide (CSP) in Streptococcus mutans is reported. Seven comC alleles encoding three distinct mature CSPs were identified among 36 geographically diverse strains, although, compared with Streptococcus pneumoniae, the amount of predicted amino acid sequence variation is low. In agreement with other studies, significant variation was found in the natural competence for DNA uptake in these strains. However, there was no correlation between the CSP genotype and the ability to transform these strains. Representative strains encoding each of the CSP variants became competent in response to synthetic CSPs of each type. Therefore, in contrast to S. pneumoniae, comC alleles in S. mutans are functionally equivalent and there is no evidence of pherotype specificity.
Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 degrees C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml-1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 microgram ml-1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.
The viral (v)-sis oncogene encodes a protein (p28sis) that is structurally homologous to platelet-derived growth factor (PDGF). We have shown that simian sarcoma virus (SSV)-transformed cells containing the v-sis oncogene release a Mr 20,000 substance that is recognized by antisera to synthetic peptide sequences contained in p28sis. Medium conditioned by SSV-transformed cells competes with 125I-labeled PDGF for specific PDGF receptor sites, initiates DNA synthesis, and stimulates tyrosine phosphorylation of the PDGF receptor when added to normal cells. When normal cells are co-cultured with SSV-transformed cells, the PDGF receptors of the normal cells are down-regulated by factors released from the transformed cells. Thus, SSV-transformed cells release material that is functionally similar to PDGF. We have used anti-phosphotyrosine antibodies to purify PDGF receptors and to detect PDGF-stimulated receptors in normal cells. SSV-transformed cells have no PDGF receptors detectable by these antibodies or by 125I-labeled PDGF binding studies. However, when SSV-transformed cells are exposed to suramin, a compound that blocks binding of PDGF to its receptors, the receptors reappear on the cell surface and within 8 hr are present at the same levels as in control cells. These "new" receptor sites can be phosphorylated in response to PDGF. Thus, the absence of PDGF receptors in SSV-transformed cells is due to down-regulation of the receptors by an autocrine mechanism that can be blocked by suramin.
Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.
Plasmid DNA transformation efficiency depends on three essential factors: 1) the optimal regime of the recipients freezing-thawing; 2) the period of the recipients competence preservation; 3) individual sensitivity of microorganisms to freezing-thawing. It is demonstrated that plasmid DNA pMB9 activity indices are of maximal value during freezing at -70 degrees C or -196 degrees C and thawing at 42 degrees C. The short period of the competence, about 15 seconds, determines the rate of its infection. In this case it was achieved by mutual freezing-thawing of bacteria and DNA pMB9. The optimal yield of transformants is obtained in the following conditions: the concentration of bacteria - 1 - 5.10(9) cells/ml, the concentration of DNA pMB9 - 0.05--0.5 mcg/ml in the reaction mixture containing 0.5--1% of bactopeptone ("Spofa") and at pH 7.4--7.6.
Uptake of DNA and genetic recombination proceeded normally in competent Streptococcus pneumoniae despite inhibition of DNA replication by 6-(p-hydroxyphenylazo)-uracil. Immediately after a brief uptake period, 68% of donor DNA label was in eclipse complex form, and 22% was in low-molecular-weight products; by the completion of integration at 10 min, 23% was integrated into the chromosome, and the rest was lost from the cell. Throughout the process, less than 1% was found as free single strands. The DNA in eclipse complex is therefore an intermediate in the integration process.
To study frequency and mechanism of homologous recombination in Streptomyces, an E. coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48. After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene (tsr) carried on pYYE04al and homologous sequences on the chromosome, S. lincolnensis YY1 and S. lincolnensis, YY2 were obtained on SMA with low thiostrepton concentration. Hybridization of chromosomal DNA samples of S. lincolnensis YY1, S. lincolnensis YY2, standard S. lincolnensis and S. lincolnensis YYc digested with SmaI with the probe of tsr gene gave signal corresponding to a fragment of 1.5 kb in the former two; Nevertheless, hybridization of chromosomal DNA digested with Hind III and Sma I using the probe of delta lacZ' gene resulted in positive fragment of 4.4 kb only in S. lincolnensis YY2. Southern hybridizations indicate that S. lincolnensis YY1 is the result of homologous exchange while S. lincolnensis YY2 comes from bomologous recombination. To prove the existence of E. coli replicon and ampicillin resistant gene on the chromosome of S. lincolnensis YY2, its DNA digested with SphI was ligated and then transformed into E. coli JM83 competent cell. Two transformants named pSLE1 grew on the plate containing ampicillin. It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes.
To simplify AdEasy system for better efficiency, we attempted some modifications on the original system. Specifically, the Pme -linearized plasmid pAdtrack was transformed into competent AdEasier-1 cells prepared from treatment with CaCl(2). The DNA contained in the identified recombinant plasmid was digested with Pac and transfected into 293 cells to package the adenovirus, followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescence microscope. Chemical transformation of the linearized plasmid into AdEasier-1 cells resulted in very high success rate (20/20) for producing positive recombinant adenovirus clones, confirming the efficiency of the modified AdEasy system in constructing recombinant adenovirus.
Electrotransformation has been used as a tool to introduce genes carried on replicative vectors in hundreds of bacterial species. In this study, the technique was used to try to obtain recombination of markers in the chromosome of the naturally transformable bacterium Streptococcus pneumoniae. Recombination was not observed even using naturally competent cultures. Both chromosomal and cloned DNA, denatured or native, were without effect. These results suggest that it is not sufficient to introduce DNA into the cell to obtain recombinants in this bacterium. The integration of markers into the chromosome in naturally competent cells must require DNA processing during entry. Electrotransformation of replicating plasmids is recA-independent but can be facilitated by a recA-dependent process. This facilitation required the induction of the natural competence machinery, probably involving partial homologous pairing.
A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.
A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.
Natural genetic transformation is a mechanism of horizontal gene transfer that is widely distributed in bacteria and requires assembly of a DNA uptake machinery. Transformable bacteria use fundamentally the same machine, which in most species is assembled only in cells that are developing competence. Competence regulation usually differs between unrelated species. Here, we examine whether related streptococci use the same competence regulatory cascade. Phylogenetic analyses of streptococcal genome sequences reveal the existence of two paralogous two-component regulatory systems, either of which might control competence. This suggests the distribution of streptococci into two groups that use competence regulatory cascades that have at least partly evolved independently. Comparison of data obtained with two transformable streptococci, Streptococcus pneumoniae and Streptococcus mutans, provides support to this suggestion.