Enhanced transcription of genes of the intracisternal A particles and B2 elements in mouse tumors

A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.     

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RIII mouse strains.

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV) produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to a greater extent, and at a lower Cot1/2 value, to the DNA of C3H mammary tumor cells than to the DNA of C3H liver cells. The 125I-labeled MMTV (C3H) 60-40S RNA was annealed to a vast excess of DNA from C3H livers, and single-stranded RNA was eluted from hydroxylapatite and recovered. This "recycled RNA" did not hybridize to the DNA of the apparently normal organs tested from normal or from mammary tumor-bearing C3H mice, but hybridized extensively to both the DNA from the C3H mammary tumor cell line and the DNA from spontaneous C3H mammary tumors. This hybridization could be competed out by the addition of unlabeled MMTV 60-70S RNA but was unaffected by the addition of unlabeled 60-70S RNA of C3H type C virus. Similar experiments were conducted with the RIII mouse strain. We therefore report on the isolation of the sequences of the RNA genomes of the MMTVs from C3H and RIII mice that are transmitted by some mechanism other than via the germ line. These studies further define the differences, via molecular hybridization, between the MMTV-S and the MMTV-L in both C3H and RIII mice.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells.

Extract of saffron (Crocus sativis) has previously been shown to inhibit colony formation and cellular DNA and RNA synthesis by HeLa cells in vitro. In order to compare the sensitivity of malignant and non-malignant cells to saffron, we examined the effect of the extract on macromolecular synthesis in three human cell lines: A549 cells (derived from a lung tumor), WI-38 cells (normal lung fibroblasts) and VA-13 cells (WI-38 cells transformed in vitro by SV40 tumor virus). We found that the malignant cells were more sensitive than the normal cells to the inhibitory effects of saffron on both DNA and RNA synthesis. There was no effect on protein synthesis in any of the cells.     

Nucleoprotein changes in plant tumor growth

Tumor cell transformation and growth were studied in a plant neoplasm, crown gall of bean, induced by Agrobacterium rubi. Ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA), histone, and total protein were estimated by microphotometry of nuclei, nucleoli, and cytoplasm in stained tissue sections. Transformation of normal cells to tumor cells was accompanied by marked increases in ribonucleoprotein content of affected tissues, reaching a maximum 2 to 3 days after inoculation with virulent bacteria. Increased DNA levels were in part associated with increased mitotic frequency, but also with progressive accumulation of nuclei in the higher DNA classes, formed by repeated DNA doubling without intervening reduction by mitosis. Some normal nuclei of the higher DNA classes (with 2, 4, or 8 times the DNA content of diploid nuclei) were reduced to diploid levels by successive cell divisions without intervening DNA synthesis. The normal relation between DNA synthesis and mitosis was thus disrupted in tumor tissue. Nevertheless, clearly defined DNA classes, as found in homologous normal tissues, were maintained in the tumor at all times.     

Homology between avian oncornavirus RNAs and DNA from several avian species.

3H-labeled 35S RNA from avian myeloblastosis virus (AMV), Rous associated virus (RAV)-0, RAV-60, RAV-61, RAV-2, or B-77(w) was hybridized with an excess of cellular DNA from different avian species, i.e., normal or leukemic chickens, normal pheasants, turkeys, Japanese quails, or ducks. Approximately two to three copies of endogenous viral DNA were estimated to be present per diploid of normal chicken cell genome. In leukemic chicken myeloblasts induced by AMV, the number of viral sequences appeared to have doubled. The hybrids formed between viral RNA and DNA from leukemic chicken cells melted with a Tm 1 to 6 C higher than that of hybrids formed between viral RNA and normal chicken cell DNA. All of the viral RNAs tested, except RAV-61, hybridized the most with DNA from AMV-infected chicken cells, followed by DNA from normal chicken cells, and then pheasant DNA. RAV-61 RNA hybridized maximally (39%) with pheasant DNA, followed by DNA from leukemic (34%), and then normal (29%) chicken cells. All viral RNAs tested hybridized little with Japanese quail DNA (2 to 5%), turkey DNA (2 to 4%), or duck DNA (1%). DNA from normal chicken cells contained only 60 to 70% of the RAV-60 genetic information, and normal pheasant cells lacked some RAV-61 DNA sequences. RAV-60 and RAV-61 genomes were more homologous to the RAV-0 genome than to the genome of RAV-2, AMV, or B-77(s). RAV-60 and RAV-61 appear to be recombinants between endogenous and exogenous viruses.     

Diacridines: bifunctional intercalators. III. Definition of the general site of action.

Electron microscopy of HeLa cells exposed to spermine diacridine shows nucleolar distortions which disappear after several days despite the persistence of the metabolic changes promoted by spermine diacridine. This compound inhibits ribosomal RNA synthesis and appears to act independently of any particular phase of the cell cycle. The DNA content of the HeLa cells remains unchanged and the cell distribution is not significantly disturbed from its normal distribution in the various phases of the cell cycle. Spermine diacridine and other diacridines inhibit primarily chain initiation but also chain elongation by DNA-directed RNA polymerase of Azotobacter vinelandii.     

A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

Nucleic acid synthesis in the normal and lobeless embryo of Ilyanassa obsoleta.

The RNA and DNA content of the normal and lobeless embryo of the Ilyanassa embryo was measured at several stages of embryogenesis. In the lobeless embryo a delay in the net accumulation of RNA and a decrease in the rate of DNA synthesis were observed. It is suggested that the failure of some of the lobe dependent organs to differentiate may be caused by a reduced rate of proliferation of determined embryonic cells.     

Estimation of cellular DNA content in cell lysates suitable for RNA isolation.

Methods presently available for the isolation of RNA are incompatible with conditions necessary for the measurement of either DNA or cell number, resulting in infrequent quantitation of messenger RNA in relation to the quantity of cells studied. In the present studies, a microfluorometric method has been modified from previous techniques to permit the quantitation of DNA in cell lysates prepared using an acid guanidinium thiocyanate-phenol (AGTP) solution from which RNA can subsequently be isolated. The lysate is incubated in alkaline EDTA, then neutralized with KH2PO4, followed by the addition of the fluorochrome bisbenzimidazole (Hoechst 33258), and measurement of fluorescence. DNA content is comparable in measurements by the present technique and by the diphenylamine method on parallel samples. DNA content per cell for human cells measured with this technique is comparable to that previously reported using other methods. The use of AGTP solution results in stability of measurable DNA in cell lysates for periods of at least 10 weeks (permitting batching of samples and retrospective measurement) and stability of fluorescence for at least 20 h after the addition of bisbenzimidazole making the timing of fluorescence measurement less critical. The technique described should permit quantitation of messenger RNA in relation to DNA (and hence indirectly to cell number) on a routine basis.     

ppGpp is the major source of growth rate control in E. coli

It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent, RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp-deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared with fast growing wild-type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp-deficient cells restores this control. We believe these findings strongly argue in favour of ppGpp and against redundant regulation of growth rate control by other factors in Escherichia coli and other enteric bacteria.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil

Reich, Melvin (The George Washington University School of Medicine, Washington, D.C.), and H. George Mandel. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar concentrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.     

Trace changes in the quantity of RNA in neurons and neuroglia of the hypothalamic nuclei of the brain following short-term intermittent exposure to cold]

Adult albino rats were kept for 2 minutes at --20 degrees C, then for 5 minutes at 25 degrees C, this intermittent cooling being repeated 15 times. One hour after the end of the cooling, the RNA quantity per cell was determined by means of visible cytospectrophotometry to find the increase in the cytoplasm of medial preoptic area neurons and in the nuclei of their glial satellite cells as well as in the nuclei of the perineuronal glial cells of mammillary bodies. Two days after the rats had stayed at the room temperature, these RNA changey bodies the RNA content decreased. 3 days later, the RNA content in the whole neuron--neuroglia unit of the preoptic area returned to normal, while in the neurons and neuroglia of mammillary bodies augmented markedly: this augmentation was found as late as 15 days after the cessation of the cooling. 30 days after the end of the cooling, the RNA quantity in the mammillary body neurons returned to normal, whiel in the neuroglia of this hypothalamic nucleus decreased even lower than the normal level. The authors discuss the problem of a so-called "vegetative memory" and of the involvement of hypothalamic neuron--neuroglia units in the metabolism tranformation dealing with a consolidation of this memory.