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PubMed1978

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.

Bacillus↗
PubMed1977

Changes in synthesis of DNA-binding proteins during the onset of transformation in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

Synthesis of cytoplasmic DNA-binding proteins was investigated after a shift from the nonpermissive to the permissive temperature in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus [ts339(RSV)]. Cells were labeled for several generations in [3H]leucine and were pulse-labeled with [35S]methionine for 1 h at the nonpermissive temperature (39 degrees C) and at the permissive temperature (33 degrees C, 5 h after shift from 39 degrees C). Proteins binding to sequential columns of double-stranded and single-stranded DNA-cellulose were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the 35S/3H ratios were obtained for each column fraction and for individual polypeptides. The protein fractions binding to single-stranded, but not double-stranded, DNA and eluting at high salt concentrations (greater than 0.60 M NaCl) showed elevated 35S/3H ratios. This indicated increased synthesis of these proteins within 5 h after the onset of transformation. The majority of the polypeptides in these fractions showed increased synthesis as a consequence of transformation. One prominent polypeptide among them constituted 0.1% of the cytosol protein and had a molecular weight of 93,000. We conclude that the synthesis of proteins binding tightly to single-stranded DNA is increased early after the onset of transformation.

Animals↗
PubMed1975

Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.

Animals↗
PubMed1979

SV40-transformed human diploid cells that remain transformed throughout their limited lifespan.

Of ninety-one subcultured foci of SV40-transformed WI-38 human diploid fibroblasts, none yielded cells that grew indefinitely, but all cells in each subculture continued to produce T antigen and to look morphologically transformed throughout their lifespan. These results are consistent with the commitment theory of fibroblast senescence, but predict that a special transformation event is necessary to accout for the rare survivors.

Antigens, Neoplasm↗
PubMed1978

Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.

Antigens, Viral↗
PubMed1979

Decreased sensitivity to interferon associated with in vitro transformation of X-ray-transformed C3H/10T1/2 cells.

We have compared the interferon sensitivity of parental 10T1/2 cells, having a phenotype characteristic of normal cells, with that of transformed cells isolated after X-irradiation of treatment with methylcholanthrene. Transformed cells were 10- to 20-fold less sensitive than the parental untransformed cells to the antiviral action of interferon and were also less sensitive to the inhibitory effect of interferon on cell multiplication. The decreased sensitivity of these cells to interferon is a useful marker of the transformed phenotype.

Animals↗
PubMed1979

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.

Animals↗
PubMed1977

Transformation in Bacillus subtilis: biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells.

Competent B. subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA. Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg2+ ions in excess of the EDTA. At 30 degrees and at 17 degrees comparable numbers of transformants are produced from cells pre-loaded with DNA at 30 degrees (after termination of uptake by the addition of DNA ase the samples were incubated at 37 degrees). However, almost no transformants are produced when cells are exposed to DNA at 17 degrees, although binding does take place then. Because DNA is taken up at 17 degrees after having loaded the cells at 30 degrees, whereas no uptake occurs after binding at 17 degrees, it is suggested that binding of DNA to the cellular surface involves at least two steps. In DNA re-extracted from cells at 17 degrees, pre-loaded with DNA at 30 degrees, little recombinant type activity is present, indicating that integration is blocked at 17 degrees. However, physico-chemical analysis of the re-extracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17 degrees. This complex has a higher buoyant density than donor-recipient complexes formed at 30 degrees.

Bacillus subtilis↗
PubMed1977

Transformation in Bacillus subtilis. Initial stages of the transformation process and their energy dependence.

NaN3 was found to inhibit transformation but not the irreversible binding of donor 3H-DNA in competent cells of the original low-transformable strain Bacillus subtilis 168 trp2. Addition of NaN3 to cells of two mutants Bacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells. The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN3, leading preferentially to lysis of the competent cells.

Azides↗
PubMed1976

Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties.

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cells cultured at the permissive (32 degrees C) and non-permissive (39 degrees C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3-4 generations in medium containing radioactive leucine (32 degrees C and 39 degrees C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (congruent to 100 000-160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growth (39 degrees C). A decrease in amount of a 120 000 molecular weight glycopeptide at 39 degrees C was the most prominent of these alterations. We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 degrees C, using lactoperoxidase-catalyzed iodination, NaBH4 reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.

Cell Fractionation↗
PubMed1978

Factors affecting the binding of polycyclic aromatic hydrocarbons to human embryo cells, and transformable and non-transformable hamster embryo cells.

The effects of various factors, including population doubling number, percent of confluence, serum concentration and storage in liquid nitrogen on the binding of several polycyclic aromatic hydrocarbons to human and hamster embryo cells were studied. The binding of 7,12-dimethylbenz[a]-anthracene (DMBA) to hamster embryo cells DNA, RNA and protein was maximal after 22 h of treatment. In contrast, binding to human embryo cell macromolecules increased for at least 55 h. Treatment of hamster embryo cells at 100% confluence resulted in much less binding than treatment at 70% confluence, whereas with human embryo cells the binding increased, or remained constant, following treatment at the greater confluence. The transforming frequency of hamster embryo cells decreases with increasing population doubling number. Accordingly, we found that the binding of DMBA to hamster embryo DNA, RNA and protein decreased approximately 100-fold between population doubling numbers 8 and 20. In transformable cell cultures, DMBA was bound to hamster embryo cell DNA to a greater extent than to RNA or protein. The binding of DMBA to nucleic acids was much greater than binding by either dibenz[a,h]anthracene (DB[a,h]A) or dibenz-[a,c]anthracene (DB[a,c]A), both of which had low binding values at all population doubling numbers tested. Therefore, the best correlation of binding with carcinogenicity and transforming activity was observed with DMBA. Storage of hamster embryo cells in liquid nitrogen did not alter their binding characteristics. Binding of all three hydrocarbons to human embryo cell nucleic acids was low during all population doubling numbers studied, while binding to cellular protein increased until population doubling number 70 and then decreased sharply.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Presence of herpesvirus-associated antigens on the surfaces of transformed, tumor, and metastatic cells and enhanced antigenicity of transformed cells using 5-iodo-2'-deoxyuridine.

Using a direct immunofluorescence technique, herpes simplex virus type 1 (HSV-1)-transformed cells and cell lines subsequently derived from tumors and metastases were examined for the presence of HSV-1-associated surface antigens. Fluorescein-labeled immunoglobulins derived from antisera were used and included anti-HSV-1 (from hamsters), sera from tumor-bearing hamsters, and antibodies prepared in rabbits against cell material shed early into the medium after infection of rabbit kidney cells with HSV-1. All immunoglobulins showed the greatest reactivity with cell lines derived from tumors and metastatic lesions. There appeared to be little or no reactivity on the surfaces of the transformed cells. Transformed cells treated with 5-iodo-2'-deoxyuridine for 48 h, however, showed an enhanced reactivity which approached that seen with the tumor-derived cell lines.

Animals↗
PubMed1979

[Spontaneous transformation characteristics of Bacillus subtilis bacteria. I. The relatively low transforming activity of spontaneously released DNA for markers located close to the origin and termination points of chromosome replication].

Relative efficiencies of spontaneous Bacillus subtilis transformation for markers placed in different areas of the cell chromosome were studied. As donor of genetic material, an untransformable strain BD224 trpC2 thr5 rec4 was used during its early log-phase. It was found that for markers placed near points of origin and termination of the chromosome replication the relative transformation efficiencies are significantly lower than those in the case of transformation with DNA extracted from the same donor cells. If a contact of spontaneously released DNA with the recipient cells was delayed for about 60 minutes ("separate" experiment) this difference proved to be less pronounced for ade16 placed near the origin, but remained practically unchanged for metB placed near the termination point. The results obtained can be explained by permanent attachment of chromosome regions, carrying "origin" and "termination" points, to a cytoplasmic membrane. During spontaneously release of cellular genetic material, "origin" and "terminal" DNA fragments carrying ade16 and metB respectively, can retain the contact with components of cell membrane. Hence, their penetration in the recipient cell and (or) participation in recombination can be violated. The first of two fragments becomes free from structurating substances more easy.

Bacillus subtilis↗
PubMed1978

[Transforming activity of plasmid R6K DNA on Serratia marcescens strain 20-10. The behavior of the plasmid in the transformants].

The authors described transformation of S. marcescens, strain 20-10, of the isolated R6K plasmide DNA. As demonstrated by centrifugation in cesium chloride gradient and electrophoresis in agarose, the plasmide was present in the transformants in the form identical to R6K in E. coli K12. Analysis of the transforming activity of R6K plasmide from Serratia and E. coli K12 strains with a complete and defective restriction system showed S. marsescens, strain 20-10, to possess specific system of restriction and modification. In studying beta-lactamase activity and Serratia and E. coli strains ampicillin and streptomycin resistance revealed differences in the phenotypical expression of the plasmide signs in the heterologous and homologous host.

DNA, Bacterial↗
PubMed1979

Spontaneous and chemically-induced transformation of mouse fibroblasts in culture. Biochemical aspects of the transformed cells.

Fibroblast cultures were established from the lung tissue of CBA T6T6 mouse embryos. Lines characterized by infinite growth transformation (MFL) were used as untreated controls till the 21st and 29th passages, respectively. After that period, an unrestrained growth transformation developed spontaneously. The cell line was then designated as STMFL. At the 8th passage of an MFL, 20-methylcholanthrene (MC) treatment was performed. The treatment resulted in a cell line (MCMFL) characterized also by unrestrained growth transformation. The nuclear protein pattern obtained by two-dimensional gel electrophoresis showed differences between STFL and MCMFL. The activity of two microsomal enzymes - aryl hydrocarbon hydroxylase and ethylmorphin demethylase - measured in the exponential growth stage of the cultures showed a decrease in the case of STMFL, compared to the MFL, and practically disappeared in the case of MCMFL.

Animals↗
PubMedDate not supplied

[Heterologous transformation: search for an explantation of the high effectiveness of Bacillus subtilis transformation achieved by using Bacillus natto DNA].

A higher efficiency of B. subtilis cells transformation by B. natto DNA is true for every concentration of DNA and is not due to the helping effect known for streptococci, strain Challis. In competition experiments for DNA uptake unlabelled B. natto and B. subtilis DNAs when added to transforming H3-DNA reduced the radioactivity of the samples by a factor of three and two, respectively. A conclusion is drawn that a higher rate of B. natto DNA penetration to the B. subtilis cells results in a higher efficiency of their transformation.

Bacillus subtilis↗
PubMed1975

Demonstration of infectious DNA in transformed cells. II. Characterization of uptake of SV40-transformed mouse cell DNA by simian cells.

The initial steps in the DNA-transfer, or transfection, method of virus rescue were characterized using primary green monkey (GMK) cells exposed to SV40-transformed mouse (SV-3T3) cell DNA in the presence of 1 mg/ml DEAE-dextran. When large amounts (10-50mug) of high molecular weight DNA (greater than 10(7) daltons) were inoculated onto 10(6) GMK cells, usually less than 1 mug became cell-associated. DNA fragmented to a size of 1-3 X 10(6) daltons was bound more efficiently by the recipient cells, but generally only 5-10 per cent of the inoculum (representing 1-4 mug) was taken up. Approximately 50 per cent of the cell-associated DNA had penetrated to a DNase-resistant state by the end of the 30-minute incubation period. The effect of the size of thr transformed cell DNA molecule on the recovery of SV40 in transfection experiments was investigated. The trend appeared to be that rescue was more efficient with the larger molecular weight samples.

Animals↗
PubMed1978

Cellular transformation and the 'morphologic phenotype' of transformed cells.

Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.

Animals↗