Precision Constraints for Three-Flavor Neutrino Oscillations from the Full MINOS+ and MINOS Dataset.

We report the final measurement of the neutrino oscillation parameters Δm_{32}^{2} and sin^{2}θ_{23} using all data from the MINOS and MINOS+ experiments. These data were collected using a total exposure of 23.76×10^{20} protons on target producing ν_{µ} and ν[over ¯]_{µ} beams and 60.75 kt yr exposure to atmospheric neutrinos. The measurement of the disappearance of ν_{µ} and the appearance of ν_{e} events between the Near and Far detectors yields |Δm_{32}^{2}|=2.40_{-0.09}^{+0.08}(2.45_{-0.08}^{+0.07})×10^{-3} eV^{2} and sin^{2}θ_{23}=0.43_{-0.04}^{+0.20}(0.42_{-0.03}^{+0.07}) at 68% C.L. for normal (inverted) hierarchy.

[Immortalization of human fibroblasts using tsA mutant of SV40 and pSV3neo plasmid]. / Immortalizatsiia fibroblastov cheloveka pod vozdeistviem tsA-mutanta SV40 i plazmidy pSV3neo.

Clones of immortalized human fibroblasts with an extended life span in culture and a capability of subloning were obtained after the infection with a temperature sensitive mutant (tsA 239) of SV40 virus and pSV3neo plasmid. As compared with the parental cells, the obtained clones exhibited increased plating efficiency, decreased doubling time, and serum dependence. We did not obtained the colony formation during cultivation of immortalized cells in semiliquid agar. This means that our cells were not completely malignant. The PCR (polymerase chain reaction)-analysis has revealed the presence of viral DNA at early passages (25th passage) after the infection by tsA SV40, and its absence after a prolonged cultivation (46th passage). PCR-analysis of the clones obtained after pSV3neo transfection has revealed the presence of gene A sequences either at early (9-15), or later (62) passages. The expression of the gene A product in cells of these clones was revealed only early passages (11 and 35). Possible mechanisms of immortal phenotype origin in human diploid cells after the action of ts-mutant and other constructions of SV40 are discussed.

[Isolation and characterization of immortalized human fibroblasts]. / Poluchenie i kharakteristika immortalizovannykh fibroblastov cheloveka.

A clone of immortalized human fibroblasts with an extended life span in culture was obtained after infection with a temperature-sensitive mutant (tsA 239) of SV40. In comparison with the parental cells, the clone exhibited an increase in the efficiency of plating, decreased doubling time, and serum dependence. No anchorage independence was observed. This means that the immortalized cells were not fully malignant, since growth in soft agar is one of the most typical features of malignancy. The PCR analysis has revealed the presence of viral DNA at early (25th) passages after infection and its absence after prolonged culturing (46th passage). These results support the data obtained after studies of T antigen expression by the indirect immunofluorescence method. No reversion to the normal phenotype was observed after transfer of the immortalized cells from the permissive temperature to the temperature restrictive for the virus (33 degrees and 39 degrees C, respectively). We deem it probable that the mutagenic effect of SV40 is realized after the hit-and-run mechanism, hence, its presence is not indispensable for the maintenance of the transformed phenotype.

[Mutagenic effect of the SV40 oncogene: induction of resistance to 6-mercaptopurine and serum independence]. / Mutagennoe deistvie onkogena SV40: induktsiia rezistentnosti k 6-merkaptopurinu i nezavisimost' ot syvorotki.

The mutagenic and transforming activity of SV40 DNA fragment, corresponding to its oncogene (the gene for large T antigen) was studied in Chinese hamster cells. After expression time of 3 to 4 days, the oncogene induced mutations of resistance to 6-mercaptopurine (6MP), while the DNA encoding the SV40 late genes, as well as DNA of Chinese hamster cells, were devoid of mutagenic activity. The value of induction ranged from 10(-4) to 10(-5). After the same expression time, the oncogene induced a typical character of oncogenic transformation - independence of serum growth factors (ser+). The value of induction of ser+ variants was somewhat higher than for resistance mutations. The study of 12 clones induced by the oncogene has shown the ser+ character to be hereditary, the expression of viral oncogene being not necessary for its maintenance. The data obtained support the hypothesis in favour of the participation of mutations of cellular genes in viral carcinogenesis.

Mutagenic effects of DNA-containing oncogenic viruses and malignant transformation of mammalian cells.

It was discovered in the 1970s that oncogenic viruses could induce gene mutations in mammalian cells. The phenomenon seems to be widespread: it was observed with all groups of DNA-containing viruses and some retroviruses. The mutagenic effects of the tested viruses at gene level are not locus specific. The viruses induce point mutations, including base substitutions, as well as deletions and insertions. The mutagenic effect of SV40 is controlled by the activity of the early A gene, which encodes the T antigen. Presumably, the process of integration creates the possibility for occurrence of mutations early after infection. Mutagenesis seems to be induced by an integrated virus, though to a much smaller extent. Virus-induced mutagenesis may be connected with an activation of the cell error-prone repair systems. The sum total of the experimental data shows that virus-induced mutagenesis and transformation are interrelated: (A) viruses, like other carcinogenes, display mutagenic activity; (B) viruses that are far removed from each other systematically, whose only similarity lay in being oncogenic and capable of integration, simultaneously showed the ability to induce gene mutations; (C) agents changing the rate of transformation also changed the rate of gene mutations: (D) The function of mutagenicity was mapped in the oncogene of SV40 (gene A); and the DNA of (E) mouse mammary carcinoma virus (MMTV) and avian leukosis virus (ALLV) induced tumors has been found to contain nucleotide sequences that transform 3T3NIH cells but do not carry any viral genetic information. Mutagenesis induced by oncogenic viruses may play a part in the multistage process of malignant transformation, though its contribution may be different in various specific cases and for different groups of viruses. Further studies of the uncommon mutagens, which viruses seem to be, may greatly increase our knowledge of the virus-cell relationship. An understanding of the extent of genetic danger inherent in viruses and live viral vaccines is necessary for practical medicine.

[Genetic nature of one of the traits of malignant cell transformation in vitro]. / Geneticheskaia priroda odnogo iz priznakov zlokachestvennoi transformatsii kletok in vitro.

The genetic events controlling the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied. A hypodiploid clone of Chinese hamster cells with normal serum requirements (49a5ser-) was used as starting material. The results of the fluctuation tests have shown that serum-independence is a random spontaneous event. Its rate of occurrence is 1-2 . 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine) revealed similar characteristics with respect to the distribution of the number of mutants in replicate cultures and the mutation rate. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the oncogenic SV40 virus significantly increased the frequency of ser+ colonies. In the majority of clones isolated in a medium with 1% serum (11 spontaneous and 7 induced by MNNG), the ser+ character proved to be stable after different periods of cultivation without selective pressure. The degree of serum-independence varied in different clones. The results suggest that the ability to grow in a medium with a low serum content originates, in most cases, from a mutation event.

The mutational origin of serum independence in Chinese hamster cells in vitro.

The genetic mechanisms determining the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied in a clone of Chinese hamster cells with normal serum requirements. The fluctuation test has shown that serum independence occurs as a random spontaneous event. Its rate of occurrence is about 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine--6MP) revealed similar characteristics with respect to the distribution of the number of mutants in replicative cultures. N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) and SV40 significantly increased the frequency of ser+ colonies. Induction was detected after an expression time of 3-4 days, which is typical of gene mutations. In 16 out of 18 ser+ clones of independent origin the ser+ character remained stable. The results suggest that the ser+ character originates in most cases from a mutation event.

The role of the transforming A gene of SV40 in the mutagenic activity of the virus.

The mutagenic activity of the tsA239 mutant of SV40 which synthetizes a defective T antigen at 40 degrees C was investigated in Chinese hamster cells under permissive and nonpermissive temperature. At 33 degrees C the virus increased the yield of 6-mercaptopurine-resistant colonies after 2 days expression time by a factor of 1.6-4 as compared with the control and raised the frequency of aberrant metaphases after the same time by a factor of 1.9-3.4. In the same experiments, with the same initially infected population of Chinese hamster cells, at 40 degrees C tsA SV40 did not induce either gene mutations or chromosome aberrations at the same early stage after infection. Presumably the activity of the A gene of SV40 is necessary not only for the transforming but also for the mutagenic effect of the virus.

The synergistic effects of SV40 and BUdR on induction of gene mutations and chromosomal aberrations in Chinese hamster cells.

The induction of chromosomal aberrations and gene mutations was studied in Chinese hamster cells after separate and combined treatment with BUdR and SV40. Separate treatment of cells with BUdR or virus infection increased the yield of chromosomal aberrations and reversions from glutamine requirement, expressed at 40 degrees C (a ts mutant), to prototrophy. The combined effect of the incorporation of BUdR into one DNA strand, and a subsequent infection by SV40 was additive as regards the percentage of aberrant metaphases. The integration of the analogue into both DNA strands followed by SV40 treatment resulted in a statistically significant increase in the frequency of aberration carrying metaphases, as compared with the frequency expected if the two agents had acted additively. The same phenomenon was detected when the frequency of reversions to glutamine independence was studied. Hence, the effect of the joint treatment by BUdR incorporated into both DNA strands and SV40 was synergistic. This is known to characterize the effect of BUdR on virus-induced transformation. Therefore, obviously the agent that enhances the malignant transformation of cells by the virus similarly modifies its mutagenic activity. The results obtained are presumed to confirm the previously advanced hypothesis that the same events following infection might control both the integration of viral DNA into the host-cell chromosome (and hence cell transformation) and virus-induced mutagenesis. The role of repair processes in the synergistic effect of BUdR and SV40 in the yield of reversions to glutamine independence is discussed.

Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells.

Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cells. In Chinese hamster cells SV40-induced chromosome aberrations were also studied. SV40 penetrated into the cells of both lines and induced synthesis of the T antigen. The efficiency of infection in Chinese hamster cells was tested additionally by their ability to form colonies in medium lacking the serum growth factor. The maximal number of cells with serum growth factor independence was observed on the first day after infection. When hamster cells had been maintained in "factor-free medium" for the first two passages after infection a sub-line was isolated, which synthesized T antigen 60 days after exposure to SV40. This was considered to be an indirect proof of the integration of viral genome into host chromosome. A significant increase in the frequency of chromosomal aberrations was detected in SV40-infected Chinese hamster cells. It was observed on the first and second days after treatment. The most numberous were the chromosome and chromatid breaks, which were distributed randomly in 5 morphological groups according to the chromosome length. SV40-induced mutations of resistance to 8-AG and 6-MP in human and Chinese hamster cells respectively were detected, when cells were plated in selective medium one to five days after infection. Induction was detected in all the 4 experiments with human cells and in 9 out of 11 experiments with Chinese hamster cells. Induction was highly significant according to the Wilcoxon test (P greater than 0.99), when the results of all experiments carried out in human and Chinese hamster cells were summarized. Resistance was stable after prolonged cultivation of 13 isolated clones under non-selective conditions. It is suggested that viral genome integration, gene mutations and chromosomal aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis is discussed.

[Mutagenesis under the influence of simian virus 40 (SV40). II. Induction of mutations for resistance to analogs of purine bases in human and Chinese hamster cells]. / Mutagenez pod vozdeistviem obez'ian'ego virusa 40 (SV40). Soobshchenie II. Induktsiia mutatsii rezistentnosti k analogam purinovykh osnovanii v kletkakh cheloveka i kitaiskogo khomiachka

Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cell lines. It is shown that SV40 penetrates into the cells studied and induces the T-antigen synthesis. The efficiency of the infection in Chinese hamster cells was tested additionally by the ability of infected cells to form colonies in the medium lacking serum growth factor. The maximal number of colony-forming cells was detected 24 hours after the infection. By culturing cells in factor-free medium a Chinese hamster cell subline was isolated, which proved to synthetize T-antigen within 60 days after the virus treatment. This is regarded as an indirect proof of the integration of viral genome into the cell genome. The increased frequency of mutants resistant to 60 mkg/ml of 8-azaquanine (human cells) and to 15 or 30 mkg/ml of 6-mercaptopurine (hamster cells) was observed on the 1-4th days following the infection. The analysis of the results of all experiments taken together revealed that the induction is highly significant according to Wilcoxon (greater than 0.99). The resistance of the isolated clones proved to be stable after the prolonged culturing under non-selective conditions. It is suggested that viral genome integration, gene mutations and chromosome aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis and tumour progression is discussed.

[Mutagenesis under the influence of simian vacuolating virus 40 (SV40). I. Induction of chromosome aberrations in Chinese hamster cells during the first days following infection]. / Mutagenez pod vozdeistviem obez'ian'ego vakuoliziruiushchego virusa 40 (sv40). Soobshchenie I. Induktsiia khromosomnykh aberratsii v kletkakh kitaiskogo khomiachka v pervye dni posle zarazheniia

Chromosome aberrations were studied in cultured chinese hamster cells on the 1--4th days after infection with Simian Virus 40. In the first and second mitoses after infection a statistically significant increase of the percentage of aberrant metaphases was observed (up to 22% as compared to 8--10% in intact cells). Already after 3 days following treatment the percentage of aberrant metaphases decreased sharply, reaching the control level. The virus induced a significant increase of the frequency of chromosome and chromatid breaks, as well as the appearance of fragments of an unknown origin. Chromosome breaks were distributed randomly among 5 morphologically distinct chromosome groups, according to the comparative length of respective chromosomes. An increase of the frequency of gaps and coiling deficiencies was also observed in virus-infected cultures. An earlier appearance of the first mitosis was observed in virus-treated cultures. It is suggested that the mutagenic effect of SV40 in the cells studied may be related to mechanisms controlling the integration of viral genomes into the cell chromosomes.