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PubMed1979

[Plasmid transformation of frozen and thawed bacteria].

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.

DNA, Bacterial↗
PubMed2004

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production.

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.

Aspergillus↗
PubMed2006

The role of estrogen in the initiation of breast cancer.

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17beta-estradiol (E(2)) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E(2), 4-OH-E(2), and 16-alpha-OH E(2)), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E(2) induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E(2) induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E(2), supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

Breast Neoplasms↗
PubMed1975

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Anti-Bacterial Agents↗
PubMed1990

Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli.

The inability of superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1)-deficient mutants of Escherichia coli to grow aerobically on minimal medium can be restored by functional complementation with a heterologous SOD-encoding sequence. Based upon this property, a phenotypic selection system has been developed for the isolation of clones containing eukaryotic SOD cDNAs. cDNA expression libraries from both Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed into a SOD-deficient E. coli strain by electroporation, and clones containing functional SODs were selected by growth on minimal medium. Analysis of these clones revealed the identity of cDNAs encoding the iron form of superoxide dismutase (FeSOD)--the first SODs of this type to be cloned from eukaryotes. The presence of this enzyme in these two divergent plant species challenges previous ideas that FeSOD is found in only a few plant families. In addition, these results show the potential for shotgun cloning of eukaryotic genes by complementation of bacterial mutants, particularly when it is combined with a highly efficient transformation method, such as electroporation.

Amino Acid Sequence↗
PubMed1977

Complementation between temperature-sensitive (ts) and host range nontransforming (hr-t) mutants of polyoma virus.

Two major classes of polyoma mutants are defective in cell transformation: early temperature-sensitive mutants of the tsA type which are defective in viral DNA synthesis and transformation at 39 degrees, but not at 32 degrees; and host range nontransforming (hr-t) mutants which fail to transform at either temperature. Mixed infection of mouse 3T3 cells by hr-t mutants and early tsA-type mutants results in enhanced growth of the tsA-type mutants at 39 degrees, indicating that the hr-t mutants can supply the early viral function required for viral DNA synthesis. The hr-t mutants also complement late is mutants which fail to produce infectious progeny at 39 degrees because of alterations in the 45,000-dalton major virion protein. Mixed infection of hamster BHK or rat Y1 cells by hr-t and tsA-type mutants results in efficient transformation at 39 degrees, indicating that the two classes of mutants can complement for transformation. No complementation is observed in pair-wise crosses among the early tsA-type mutants alone. The tsA-type mutants are located in the distal portion of the early region of the polyoma genome [Miller, L. K., and Fried, M. (1976) J. Virol. 18, 824-832]. The hr-t mutants are located in the proximal portion [Feunteun, J., Sompayrac, L., Fluck, M., and Benjamin, T. (1976) Proc. Nat. Acad. Sci. USA]. These results suggest that the early region of the polyoma genome is divided into two functional regions which can complement for transformation. The ts3 mutant of polyoma is located in the proximal portion of the late region.

Cell Line↗
PubMed1992

Bacteriophage P1 gene 10 is expressed from a promoter-operator sequence controlled by C1 and Bof proteins.

Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences. In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified. Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter. Production of gp10-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth. The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions. Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: C1 repressor and Bof modulator. Both proteins are necessary for complete repression of gene 10 expression during lysogeny. Under conditions that did not ensure repression by C1 and Bof, the expression of gp10-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability. Results of in vitro DNA-binding studies confirmed that C1 binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94. Although Bof alone does not bind to DNA, together with C1 it increases the efficiency of the repressor-operator interaction. These results are in line with the idea that gp10 plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1.

Base Sequence↗
PubMed1987

Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication.

The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.

Bacterial Proteins↗
PubMed1995

An improved Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation.

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7.9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10(5) cfu micrograms-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus, it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.

DNA, Bacterial↗
PubMed1992

Oncogenic activation of murine mos protein kinase by DNA rearrangement of its N-terminal coding region.

An activated c-mos oncogene was detected by DNA transfection assay of hamster SHOK cells with DNAs from X-ray-induced mouse osteosarcoma. It was molecularly cloned by the cosmid rescue method and found to form transformed foci of SHOK cells. Genomic DNA sequencing revealed that in this oncogene the N-terminal coding region of the mouse proto-mos gene was deleted and replaced by a hamster-derived sequence in the primary transformant, suggesting that activation was due to the rearrangement during transfection. The gene product was about 37 kDa and was immunoprecipitated with anti-mos antibody from a lysate of a SHOK cell transfectant. This truncated mos (t-mos) gene transformed SHOK cells more effectively than v-mos. A chimeric gene construct of this hamster-derived upstream sequence and normal mouse c-mos also transformed SHOK cells at a lower level, whereas neither t-mos nor the chimeric c-mos gene transformed NIH3T3 cells appreciably. The high transforming efficiency of t-mos in SHOK cells was due not only to truncation of the coding region but also to its integration under a putative promoter sequence derived from the hamster genome. This is the first report of detection of an activated c-mos gene by DNA transfection assay.

Amino Acid Sequence↗
PubMed2006

Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegans.

A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed.

Actinomycetales↗
PubMed1992

In vitro evolution of cisplatin/DNA monoadducts into diadducts is dependent upon superhelical density.

DNA binding of antitumor platinum(II) compounds accounts for cellular toxicity. Binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to DNA involves the transient presence of monoadducts which evolve in a second phase into difunctional lesions which are far more toxic than the monoadducts. Temporal control of the monoadducts half-live is at least dependent upon the chemical nature of the cis-platinum derivative and the secondary structure of DNA. The effect of the degree of DNA superhelicity on the binding of cis-platinum derivatives as well as on the evolution of monofunctional adducts has been addressed on plasmid DNA. The rate of platination was not affected by the degree of DNA superhelicity. Similarly, when the evolution of the lesions was complete, no variation of toxicity was found with different populations of topoisomers, as determined by bacterial transformation efficiency. In contrast, when the kinetic of difunctional lesions formation was controlled in vitro, we observed a higher rate of formation on a supercoiled plasmid by comparison with a relaxed one. This result suggests that platinum-DNA adduct toxicity could be modulated by the topology of the chromosome.

Cisplatin↗
PubMed2003

The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.

Blotting, Northern↗
PubMed2003

Agrobacterium tumefaciens-mediated genetic transformation of the cardenolide-producing plant Digitalis minor L.

A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4 %) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD550 of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integration was obtained by PCR, growth on media selective for nptII or bar genes, and expression of the gusA gene. Southern hybridisation, performed in six plants, provided information about the number of inserts. More than 200 transgenic plants were recovered from 65 independent explants. Thirty of these plants were successfully established in soil. This is the first report on transgenic Digitalis spp plants using an A. tumefaciens-mediated leaf disc transformation procedure.

Agrobacterium tumefaciens↗
PubMed2000

Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method.

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

Arabidopsis↗
PubMed1991

Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor.

The transforming proteins of several DNA tumour viruses, including adenovirus E1a and simian virus 40 large T antigen, complex with the retinoblastoma (Rb) tumour-suppressor gene product. This requires regions in these viral proteins necessary for transformation and is thought to inactivate the growth-suppressing properties of the Rb protein by disrupting its interaction with cellular targets. Indeed, regions of Rb required to form a complex with E1a and large T antigen are often mutated in transformed cells. The level at which the Rb protein regulates proliferation is unknown, although one possibility is transcription. We have previously characterized a sequence-specific transcription factor, DRTF1, the activity of which is downregulated as embryonal carcinoma stem cells differentiate. DRTF1 is found in several discrete protein complexes (a, b and c) which are of different sizes but have the same DNA specificity. We now show that one of these also contains the Rb protein and, further, that the adenovirus E1a protein causes the dissociation of the Rb protein from this complex. This requires conserved regions 1 and 2 of E1a that are known to be required for efficient transformation. These results demonstrate that the Rb protein forms a complex with a DNA-bound transcription factor, and suggests that the Rb protein might act by regulating transcription.

Adenovirus Early Proteins↗
PubMed2004

Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes.

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5' flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.

5' Flanking Region↗
PubMed1973

Temperature-sensitive initiation of chromosome replication in a mutant of Bacillus subtilis.

A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.

Amino Acids↗