DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

DNA-mediated transformation of the basidiomycete Coprinus cinereus.

We have developed a simple and efficient transformation system for the agaric fungus, Coprinus cinereus. Protoplasts were prepared from asexual spores that harbor one or two mutations in the structural gene for tryptophan synthetase. The protoplasts can be stably transformed using the cloned Coprinus gene at a frequency of 1 in 10(4) viable protoplasts. A variety of molecular events accompanies the formation of stable transformants, including insertion of the transforming DNA at the homologous locus. The transforming DNA is stable through cell division, mating, fruiting body formation, and meiosis.     

Fungal transformation of fluoranthene.

The fungus Cunninghamella elegans ATCC 36112 metabolized approximately 80% of the 3-14C-labeled fluoranthene (FA) added within 72 h of incubation. C. elegans metabolized FA to trans-2,3-dihydroxy-2,3-dihydrofluoranthene (trans-2,3-dihydrodiol), 8- and 9-hydroxyfluoranthene trans-2,3-dihydrodiol, 3-fluoranthene-beta-glucopyranoside, and 3-(8-hydroxyfluoranthene)-beta-glucopyranoside. These metabolites were separated by thin-layer and reversed-phase high-performance liquid chromatography and identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques. The major pathway involved hydroxylation to form a glucoside conjugate of 3-hydroxyfluoranthene and a glucoside conjugate of 3,8-dihydroxyfluoranthene which together accounted for 52% of the total ethyl acetate-soluble metabolites. C. elegans initially metabolized FA in the 2,3 position to form fluoranthene trans-2,3-dihydrodiol, which has previously been shown to be a biologically active compound in mammalian and bacterial genotoxicity tests. However, C. elegans formed predominantly glucoside conjugates of the phenolic derivatives of FA, which suggests that this fungus has the potential to detoxify FA.     

Microbial transformation of kepone.

Pseudomonas aeruginosa strain KO3 and a mixed aerobic enrichment culture, isolated from sewage sludge lagoon water, were found to aerobically transform the chlorinated insecticide Kepone, yielding monohydro-Kepone. Identification of the product was confirmed by gas chromatography and electron impact mass spectrometry. The mixed culture and P. aeruginosa strain KO3 produced about 4 and 16%, respectively, dihydro-Kepone, determined by cochromatography using authentic standards. Reduced amounts of monohydro-Kepone, compared with the mixed and pure cultures, were produced by James River sediment microorganisms. Kepone was not utilized as a sole carbon or energy source by any of the bacteria or mixed cultures examined in this study.     

Interspecies transformation in Bacillus: mechanism of heterologous intergenote transformation.

Bacillus subtilis-Bacillus globigii hybrids were made by integration of the B. globigii aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformation by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 X 10(6) to 40 X 10(6) daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigii DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences of both flanks of the intergenote.     

Stable nuclear transformation of the diatom Chaetoceros sp.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 10⁸ cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.     

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.     

A kinetic analysis of the estrogen receptor transformation.

The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptor's subunits reflect conformational changes involved in receptor activation.     

Structural transformation of the nuclear matrix in situ.

A striking difference in structure can be revealed between the nuclear matrix isolated from the macronuclei of normal Tetrahymena cells and the nuclear matrix of cells pretreated with 50 μg/ml actinomycin D for 2 h. The latter matrix-type shows residual giant fusion-nucleoli, which are formed in the macronuclei of actinomycin-treated cells. Both nuclear matrix types, however, exhibit an identical qualitative protein pattern in 15% SDS-gel electrophoretograms, with only minor changes in the staining properties of a few peptides. These data support the view that the nuclear matrix represents the residual equivalent to an in situ existing nuclear protein framework, which must be regarded not as a rigid, but rather as a dynamic flexible framework.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.