Synthesis of ribonucleic acid in baby-hamster kidney cells in the presence of actinomycin D.
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ABSTRACT:
SUBMITTER: Martin SJ
PROVIDER: S-EPMC1198416 | biostudies-other | 1967 Dec
REPOSITORIES: biostudies-other
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Project description:1. The sucrose-gradient pattern of (32)P-labelled RNA synthesized in actinomycintreated baby-hamster kidney cells infected with foot-and-mouth-disease virus depends greatly on the period of labelling. 2. Fractions are formed in infected cells that sediment at 12-20s and have the same base composition as similar fractions found in non-infected cells that have been treated with actinomycin. 3. In the presence of guanidine, which completely inhibits viral RNA synthesis, these fractions are labelled to a greater extent than in non-infected cells.
| S-EPMC1198417 | biostudies-other
Project description:1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol-EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4 degrees or heating at 95 degrees followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95 degrees caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95 degrees caused some aggregation of 18s material with the 28s fraction.
| S-EPMC1270179 | biostudies-other
Project description:The role of polyamines in macromolecular synthesis has been studied using the synthesis of Semliki-Forest virus (SF virus) in normal and alpha-difluoromethylornithine-treated baby-hamster kidney (BHK21) cells as a model system. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, the rate-limiting enzymes in polyamine biosynthesis, decreased rapidly in mock- and SF-virus-infected cells, indicating that virus production in BHK21 cells was not dependent on polyamines formed after infection. A prolonged treatment of BHK21 cells with alpha-difluoro-methylornithine, a specific inhibitor of polyamine synthesis, resulted in a marked inhibition of the initial rate of virus production, which appeared 72 h after the beginning of the treatment. This inhibition was reversed by putrescine, spermidine and spermine, and at last partially by several other diamines and polyamine homologues. Polyamine-depletion also markedly reduced viral RNA polymerase activity in SF-virus infected cells. Addition of spermidine to the culture medium rapidly increased viral RNA polymerase activity in the inhibitor-treated cells but had no effect on the enzyme activity when added directly to the assay mixture. The results indicated that polyamines are needed for maximum initial rate of SF-virus replication and suggest that the inhibition of virus production in polyamine-depleted cells is at least partly due to malfunction of the protein-synthetic machinery of the host cell.
| S-EPMC1158556 | biostudies-other
Project description:A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.
| S-EPMC1186561 | biostudies-other
Project description:Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.
| S-EPMC1151098 | biostudies-other
Project description:RNAseq analysis identified 837 differential gene expressions (DEGs) with 423 up-regulated and 414 down-regulated after transfecting STYK1/NOK into BHK21 cells. GEO and KEGG enrichment analysis indicated that several entries such as ribosome, aging related diseases, oxidative phosphorylation and apoptosis played major roles during this cellular process. PPI analysis showed that four top down-regulated proteins (TOMM7, Uba52, Rpl28 and XP_005077076.1 (ribosomal protein S29)) localized within the highly interaction region might play key roles.
2021-04-15 | GSE172045 | GEO
Project description:The segregation of human cathepsin D, studied in baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA and compared with that of hamster cathepsin D in the same cells, showed that, in cells that expressed human cathepsin D at a low rate, most of the enzyme remained intracellular. In contrast, when the enzyme was expressed at a high rate, most was secreted. The segregation was examined with an anti-(human cathepsin D) antibody that reacted with the human enzyme exclusively and an anti-(rat cathepsin D) antibody that reacted with both enzymes. In one protocol the cells were metabolically labelled and the two antibodies were used in sequence to precipitate the enzymes from extracts of cells and medium. High expression of the human enzyme did not interfere with the segregation of hamster cathepsin D. In another protocol the activity of cathepsin D in cells and medium was measured before and after titration with anti-(human cathepsin D) antiserum. Human cathepsin D was found predominantly in the medium, and hamster cathepsin D mainly in the cells. In the presence of 10 mM-NH4Cl the intracellular segregation of hamster cathepsin D was strongly inhibited, while the segregation of human cathepsin D was only slightly diminished. In BHK cells, at least two systems participate in the sorting of the two cathepsins, one of them being rather insensitive to NH4Cl.
| S-EPMC1149854 | biostudies-other
Project description:Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.
| S-EPMC8177424 | biostudies-literature
Project description:In medium and in homogenates from baby-hamster kidney cells (BHK) transfected with human cathepsin D cDNA, an elevated activity of cathepsin D was found as compared to non-transfected cells. The elevated activity was removed by titrating the homogenates with an anti-(human cathepsin D) antibody. Metabolic labelling and immunoprecipitation revealed that, in the transfected cells, human cathepsin D was synthesized as a 53-kDa precursor indistinguishable from that found in human cells. A portion of the precursor was secreted and the remainder was processed to intermediate and mature chains within a few hours of synthesis. The precursor that was released from the transfected cells had a slightly smaller apparent size than that from cultured human fibroblasts. This difference was abrogated when the precursors were treated with glycopeptidase F. In the intracellular small chain a difference was observed in the size of carbohydrate chains that were cleavable with endo-beta-N-acetylglucosaminidase H. Sequence analysis of the N-termini of mature intracellular cathepsin D indicated a N-terminal trimming in both large and small chains from both human and transfected hamster cells. The proteolytic maturation of human cathepsin D in BHK cells closely resembles that in human cells, whereas a portion of the carbohydrate side chains is processed differently. The trimming of the N-termini in mature cathepsin D is proposed to be a part of the maturation and aging of this protein.
| S-EPMC1149853 | biostudies-other
Project description:5'-Methylthioadenosine was taken up and immediately metabolized further by cultured baby-hamster kidney cells during the exponential phase of growth. The adenine moiety supplied the purine-nucleotide pool via the salvage pathway and was efficiently incorporated into nucleic acids. Catabolites of methylthioadenosine excreted by the cells included adenine, purinic compounds and metabolites of the ribose portion. 5'-Methylthiotubercidin had no significant effect on the cellular metabolism of methyl-thioadenosine, but greatly inhibited its uptake. erythro-9-(2-Hydroxy-3-nonyl)adenine had no effect on the uptake, but markedly interfered with the further utilization of methylthioadenosine after cleavage in the cells.
| S-EPMC1158423 | biostudies-other