656 results on '"HeLa Cells analysis"'
Search Results
2. Human papillomavirus type 18 in conjunctival intraepithelial neoplasia.
- Author
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Lauer SA, Malter JS, and Meier JR
- Subjects
- Autoradiography, Cell Line, Cervix Uteri analysis, Cervix Uteri cytology, DNA, Viral analysis, Electrophoresis, Agar Gel, Female, HeLa Cells analysis, Humans, Papillomaviridae genetics, Polymerase Chain Reaction, Conjunctival Neoplasms microbiology, Papillomaviridae isolation & purification
- Abstract
Human papillomaviruses are oncogenic viruses that have been found in a variety of epithelial neoplasias. We sought to confirm their presence in conjunctival intraepithelial neoplasia. Five tumors were studied with a polymerase chain-reaction assay designed to detect the E6 region of human papillomavirus types 16 and 18. Human papillomavirus type-16 DNA was found in four of the five tumors, including two tumors that contained both type-16 and type-18 DNA. Viral DNA was not present in the fifth tumor.
- Published
- 1990
- Full Text
- View/download PDF
3. Resolution of factors required for the initiation of transcription by yeast RNA polymerase II.
- Author
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Flanagan PM, Kelleher RJ, Feaver WJ, Lue NF, LaPointe JW, and Kornberg RD
- Subjects
- Cell Nucleus enzymology, Escherichia coli metabolism, HeLa Cells analysis, Saccharomyces cerevisiae ultrastructure, Transcription Factor TFIIA, Transcription Factor TFIID, Transcription Factors isolation & purification, RNA Polymerase II metabolism, Saccharomyces cerevisiae enzymology, Transcription Factors pharmacology, Transcription, Genetic
- Abstract
Fractionation of a yeast nuclear extract reveals at least four factors required in addition to RNA polymerase II for accurate initiation of transcription. One of these factors can be replaced by HeLa transcription factor IID or by its yeast counterpart expressed in Escherichia coli. Each of the remaining three factors can be replaced by a fraction from yeast whole cell extract, facilitating further purification of the factors.
- Published
- 1990
4. Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.
- Author
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Spector DH, Wade E, Wright DA, Koval V, Clark C, Jaquish D, and Spector SA
- Subjects
- Animals, CD4 Antigens analysis, CD4 Antigens genetics, Cell Transformation, Viral, Cells, Cultured, Fibroblasts, HIV classification, HIV genetics, HeLa Cells analysis, Humans, Male, Mice, Nucleic Acid Hybridization, Poly A genetics, Poly A isolation & purification, RNA genetics, RNA isolation & purification, RNA, Messenger, RNA, Viral analysis, RNA, Viral genetics, Retroviridae genetics, Retroviridae physiology, Skin, HIV physiology
- Abstract
One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.
- Published
- 1990
- Full Text
- View/download PDF
5. Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus.
- Author
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Mole-Bajer J, Bajer AS, Zinkowski RP, Balczon RD, and Brinkley BR
- Subjects
- Blotting, Western, Cell Cycle, Cell Nucleus analysis, Chromosomes ultrastructure, Electrophoresis, Polyacrylamide Gel, Fluorescent Antibody Technique, HeLa Cells analysis, Humans, Molecular Weight, Nuclear Proteins analysis, Nuclear Proteins immunology, Nuclear Proteins isolation & purification, Plant Cells, Plants immunology, Autoantibodies immunology, Chromosomes analysis, Plants genetics, Scleroderma, Systemic immunology
- Abstract
Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations of prometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/kinetochore components throughout evolution.
- Published
- 1990
- Full Text
- View/download PDF
6. Human Ro ribonucleoprotein particles: characterization of native structure and stable association with the La polypeptide.
- Author
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Boire G and Craft J
- Subjects
- Autoantigens immunology, Chromatography, High Pressure Liquid, HeLa Cells analysis, Humans, Molecular Weight, Osmolar Concentration, Precipitin Tests, RNA analysis, SS-B Antigen, Autoantigens analysis, Autoantigens isolation & purification, RNA, Small Cytoplasmic, Ribonucleoproteins analysis
- Abstract
Anti-Ro autoantibodies, found in sera of patients with systemic lupus erythematosus, Sjogren's syndrome, and related diseases, target the Ro ribonucleoprotein particles (RNPs). Although the polypeptide and RNA components of the Ro RNPs have been characterized, much less is known about the native structure of these particles. We have now characterized by biochemical techniques intact Ro ribonucleoprotein particles from cultured HeLa cells. These particles segregated in three discrete subpopulations with characteristic physicochemical properties: one containing hY5 RNA (RohY5 particles), one containing only hY4 RNA (RohY4 particles) and one with hY1, hY3, and hY4 RNAs (RohY1-hY4 particles). The RohY5 particles were purified free of contaminating ribonucleoproteins; both the La and the 60-kD Ro polypeptides were stable components of this portion of the Ro RNPs. The La RNPs co-purified with the RohY4 particles and contaminated the RohY1-hY4 RNPs. The stable association between the La and the 60-kD Ro polypeptides provides a potential macromolecular target for the linked set of anti-Ro and anti-La antibodies, and suggests a possible functional association of these polypeptides.
- Published
- 1990
- Full Text
- View/download PDF
7. Bacteriophage T4 gene 32 protein shares antigen determinants with reconstituted HeLa hnRNP proteins in western blots.
- Author
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Schenkel J and Hovemann B
- Subjects
- Blotting, Western, DNA-Binding Proteins isolation & purification, Escherichia coli genetics, HeLa Cells analysis, Heterogeneous-Nuclear Ribonucleoproteins, Humans, Immune Sera, Molecular Weight, Ribonucleoproteins isolation & purification, Viral Proteins isolation & purification, DNA-Binding Proteins immunology, Epitopes analysis, Ribonucleoproteins immunology, T-Phages genetics, Viral Proteins immunology
- Abstract
A polyclonal antiserum against purified bacteriophage T4 gene 32 protein was raised in rabbits. In Western blots it detected a number of SDS-PAGE separated nuclear and ribosomal proteins of HeLa cells. Using a renaturing blotting system, however, larger hnRNP proteins ranging between 66.000 and 82.000 Da preferentially reacted with this antiserum. In addition hnRNP group A core proteins were detected to a minor extent. Nucleic acid binding proteins like histones or ribosomal proteins were not stained by this antibody after renaturation.
- Published
- 1990
- Full Text
- View/download PDF
8. Effects of osmotic manipulation of intracellular hydration of HeLa S-3 cells on their proton NMR relaxation times.
- Author
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Wheatley DN, Rimmington JE, and Foster MA
- Subjects
- HeLa Cells analysis, HeLa Cells ultrastructure, Humans, Hypertonic Solutions, Hypotonic Solutions, Isotonic Solutions, Microscopy, Electron, Osmolar Concentration, Time Factors, Body Fluids physiology, HeLa Cells physiology, Intracellular Fluid physiology, Magnetic Resonance Spectroscopy methods
- Abstract
Pellets of HeLa from suspension cultured cells in isotonic medium (300 mosmolar) were introduced into a Bruker CXP100 NMR spectrophotometer at 80 mHz within 5 min of the start of centrifugation. T1 and T2 times were measured within a total elapsed time of 20-25 min at 80 mHz and 37 degrees C, and averaged 1430 msec and 120 msec, respectively. Extrapolation to zero extracellular space gave a corrected T1 of 1370 msec. For cells collected after 10 min in hypotonic medium (down to 30 mosmolar) increased proton density correlated well with increased cell water content, but relaxation times did not rise in proportion to that predicted for the entry of "bulk" water (T1 of 4700 msec), except when swelling approached lysis point. Cells partially dehydrated by 10 min in hypertonic medium of up to 1500 mosmolar have also been analyzed, but once again the shortening of T1 was not proportional to the loss of "free" (bulk phase) water. At the upper limit of hypertonic treatment, lacunae or vacuoles of a watery nature separated within the cytomatrix, preventing maximum dehydration. The relationship of cell water to T1 is complex over the whole range of tonicity that HeLa S-3 cells tolerate. The data indicate, however, that hypotonically induced water probably has an average T1 time considerably lower than bulk phase water. In contrast, raising the total extracellular volume with medium had precisely the predicted effect on T1 time, further strengthening the case that water taken up by cell acquires a shorter T1 time. Cells adapting to hypotonic conditions oscillated in size and water content over 2-3 hr before returning to near their initial volume. Under these circumstances, T1 oscillated in the same way but with a reduced amplitude, consistent with the above findings.
- Published
- 1990
- Full Text
- View/download PDF
9. Immuno affinity purification and characterization of a stable U4/U5/U6 multip-snRNP complex from HeLa nuclear extracts.
- Author
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Behrens SE and Lührmann R
- Subjects
- Chromatography, Affinity, HeLa Cells analysis, Humans, Immunosorbent Techniques, RNA, Small Nuclear isolation & purification, Ribonucleoproteins, Small Nuclear, Ribonucleoproteins isolation & purification
- Published
- 1990
- Full Text
- View/download PDF
10. The Th ribonucleoprotein particle: association with the RNA processing enzymes RNase MRP and RNase P.
- Author
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Craft J and Gold H
- Subjects
- Cell Nucleolus metabolism, Cell Nucleus metabolism, HeLa Cells analysis, Humans, RNA Processing, Post-Transcriptional, Ribonuclease P, Ribonucleoproteins, Small Nuclear, Endoribonucleases metabolism, RNA metabolism, Ribonucleoproteins metabolism
- Published
- 1990
- Full Text
- View/download PDF
11. Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method.
- Author
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Hougaard DM and Larsson LI
- Subjects
- Breast Neoplasms analysis, Breast Neoplasms pathology, Cytophotometry methods, Fluorescamine, Fluorescence, Formaldehyde, HeLa Cells analysis, HeLa Cells pathology, Humans, Methods, Tumor Cells, Cultured, o-Phthalaldehyde, Spermidine analysis, Spermine analysis
- Abstract
Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.
- Published
- 1990
- Full Text
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12. Affinity chromatography of HeLa splicing extracts on m7 GTP-sepharose and photoaffinity-labeling of cap-binding proteins with gamma-[(4-benzoylphenyl) methylamido]-7-methyl-guanosine-5'-triphosphate.
- Author
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Thalmann E, Blaas D, and Kuechler E
- Subjects
- Chromatography, Affinity, Humans, RNA Cap-Binding Proteins, RNA Splicing, Affinity Labels, Carrier Proteins isolation & purification, Guanosine Triphosphate analogs & derivatives, HeLa Cells analysis
- Published
- 1990
- Full Text
- View/download PDF
13. Components involved in nuclear pre-mRNA splicing.
- Author
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Krämer A, Utans U, Keller W, and Lamond A
- Subjects
- HeLa Cells analysis, Humans, Ribonucleoproteins, Small Nuclear, RNA Precursors metabolism, RNA Splicing, RNA, Messenger metabolism, RNA, Small Nuclear physiology, Ribonucleoproteins physiology
- Published
- 1990
- Full Text
- View/download PDF
14. Distribution of repetitive and nonrepetivite sequence transcripts in HeLa mRNA.
- Author
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Klein WH, Murphy W, Attardi G, Britten RJ, and Davidson EH
- Subjects
- Adenosine, Base Sequence, Chromatography, Female, Genetic Code, HeLa Cells analysis, Humans, Hydroxyapatites, Kinetics, Polynucleotides, DNA, HeLa Cells metabolism, Nucleic Acid Hybridization, RNA, Messenger analysis, RNA, Neoplasm analysis, Transcription, Genetic
- Abstract
Polyadenylated messenger RNA extracted from HeLa cells was hybridized with a mass excess of HeLa DNA. The kinetics of the hybridization reaction demonstrated that most of the messenger RNA is transcribed from nonrepetitive DNA. The amount of messenger RNA hybridized to DNA was measured both with and without prior RNase treatment. Comparison of the results indicates that within the limits of detection, HeLa messenger RNA does not contain repetitive sequence elements covalently linked to nonrepetitive sequence transcripts. However, a small fraction of the HeLa messenger RNA preparation is transcribed entirely from repetitive DNA sequences. This fraction represents about 6% of the total polyadenylated messenger RNA preparation.
- Published
- 1974
- Full Text
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15. Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation.
- Author
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Duncan R and Hershey JW
- Subjects
- Centrifugation, Density Gradient, Electrophoresis, Polyacrylamide Gel, Heat-Shock Proteins biosynthesis, Humans, Immunosorbent Techniques, Peptide Initiation Factors biosynthesis, HeLa Cells analysis, Hot Temperature, Protein Biosynthesis
- Abstract
Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.
- Published
- 1984
16. Cytokeratins in human basal and squamous cell carcinomas: biochemical, immunohistological findings and comparisons with normal epithelia.
- Author
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Viac J, Reano A, and Thivolet J
- Subjects
- Animals, Epithelium analysis, Fluorescent Antibody Technique, HeLa Cells analysis, Humans, Intestines analysis, Mouth Mucosa analysis, Rabbits, Rats, Carcinoma, Basal Cell analysis, Carcinoma, Squamous Cell analysis, Keratins analysis, Skin analysis
- Abstract
The nature of epithelial cell cytokeratins from epidermal basal cell carcinomas (BCC) (8 cases) and squamous cell carcinomas (SCC) (5 cases) was investigated by biochemical and immunological analysis. Cytokeratin proteins were extracted with high salt buffer and triton X 100 and were comparatively analyzed by SDS (sodium dodecyl sulphate) polyacrylamide gel electrophoresis. Both types of tumor showed either an absence or a very low amount (5% of the total protein) of the major protein band (MW 67000) present in normal human epidermis. This correlated well with results of immunolabelling showing that 67000 keratin antisera, only reacted with some dyskeratotic cells in sections of these tumors. Gel electrophoresis showed in BCC and SCC, three distinct groups of predominant polypeptide bands of apparent relative MW: (1) 60-62000 (2) 54-56000 and (3) 49000, representing respectively about 43.0%, 31.0% and 20.4% of the total proteins. Antibodies raised in animals against polypeptide bands C1 (MW 62000), C2 (MW 56000) and C3 (MW 49000) from SCC, strongly labelled (indirect immunofluorescence) all malignant cells present in the 2 kinds of tumors. These antisera showed a preferential reaction with the basal epithelial cells, in sections of human and animal epidermis and mucosa thus, suggesting numerous common antigenic determinants between epithelial cells from diverse origins. On the other hand, strong differences between mucosal and epidermal upper layers were noted with C1, C2, C3 and 67000 antisera. These results are further evidence for the existence of different pathways of keratinization in epidermis and mucosa.
- Published
- 1982
- Full Text
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17. Preservation of RNA and DNA in samples from HeLa suspension cultures.
- Author
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Thilly WG, Arkin DI, and Wogan GN
- Subjects
- Deoxyribonucleases, Drug Stability, Freezing, Humans, Osmolar Concentration, Refrigeration, Ribonucleases, Spectrophotometry, Ultraviolet, Sulfates, Temperature, Time Factors, Zinc, DNA, Neoplasm analysis, HeLa Cells analysis, RNA, Neoplasm analysis
- Published
- 1974
- Full Text
- View/download PDF
18. Sequence analysis and precise mapping of the 3' ends of HeLa cell mitochondrial ribosomal RNAs.
- Author
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Dubin DT, Montoya J, Timko KD, and Attardi G
- Subjects
- Base Sequence, Chromatography, DEAE-Cellulose, DNA, Mitochondrial, Electrophoresis, Paper, HeLa Cells analysis, Humans, Oligonucleotides analysis, Ribonucleases, Ribonucleotides analysis, Mitochondria analysis, RNA, Ribosomal
- Published
- 1982
- Full Text
- View/download PDF
19. A 52-kD protein is a novel component of the SS-A/Ro antigenic particle.
- Author
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Ben-Chetrit E, Chan EK, Sullivan KF, and Tan EM
- Subjects
- Autoantibodies immunology, Autoantigens immunology, Cross Reactions, HeLa Cells analysis, Humans, Proteins immunology, RNA, Small Nuclear immunology, SS-B Antigen, Autoantigens analysis, Proteins isolation & purification, RNA, Small Cytoplasmic, Ribonucleoproteins, Sjogren's Syndrome immunology
- Abstract
Anti-SS-A/Ro autoantibodies are found in the sera of patients with Sjogren's syndrome (SS) and SLE. In the course of analyzing 61 SS patients for their autoantibody profiles, we found that 42 were positive for anti-SS-A by double diffusion in agarose and demonstrated precipitin lines identical to that produced by a prototype anti-SS-A serum. Further analysis of these SS-A antibody-positive sera by Western blotting of cell extracts revealed that 21 sera reacted with two proteins of 60 and 52 kD, 13 sera reacted with 52-kD protein, two detected only 60 kD, while six were nonreactive. Affinity-purified anti-60-kD and anti-52-kD antibodies reacted exclusively with their corresponding antigens. Partial proteolysis of these proteins did not reveal common degradation fragments. Thus the 52- and 60-kD proteins were found to be antigenically and apparently structurally distinct from each other. They were also distinct from 48-kD SS-B/La protein. In immunoprecipitation using labeled cell extracts, affinity-purified anti-52-kD antibodies brought down the 52-kD protein as well as the 60-kD band. In [32P]orthophosphate-labeled HeLa cell extract both antibodies precipitated the same spectrum of small RNAs (hYl-5). In indirect immunofluorescence, anti-52-kD and anti-60-kD antibodies immunolocalized in similar subcellular structures and showed similar punctate nuclear staining patterns. Western blot analysis revealed that both proteins were present in lymphocytic as well as epithelial human cell lines tested. The data above define a new antigen of 52 kD which is another component of the SS-A particle and is associated in complex formation with the previously reported 60-kD protein.
- Published
- 1988
- Full Text
- View/download PDF
20. Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates.
- Author
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North TW, Bestwick RK, and Mathews CK
- Subjects
- DNA-Directed DNA Polymerase metabolism, HeLa Cells analysis, HeLa Cells enzymology, Humans, Kinetics, Methods, Nucleoside-Diphosphate Kinase antagonists & inhibitors, Phosphotransferases antagonists & inhibitors, Deoxyribonucleotides analysis, Methyltransferases analysis, Nucleoside-Diphosphate Kinase analysis, Phosphotransferases analysis, Phosphotransferases (Phosphate Group Acceptor), Thymidylate Synthase analysis
- Abstract
Several enzymes that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates (dNTP's) are present as contaminants when nucleotides are extracted from HeLa cells with 60% methanol. These activities include a nuclease, nucleoside diphosphokinase, and deoxyribonucleoside monophosphokinases which phosphorylate dAMP, dGMP, and dCMP. Collectively, these enzymes are able to degrade and reutilize the DNA template which is used together with DNA polymerase for dNTP assays. This process introduces large errors when dNTP assays are performed in this manner. Attempts to block the enzymatic conversion of deoxyribonucleoside diphosphates to triphosphates by inhibition of nucleoside diphosphokinase were unsuccessful because of the inability to block completely the kinase activity. Acid extraction of nucleotides also results in the presence of an activity that interferes with the enzymatic dNTP assay. The error introduced by this interfering activity is much smaller than that arising from the enzymes present in methanol extracts. All of these interfering activities are removed when cells are first extracted with 60% methanol and the resulting extract is subsequently treated with perchloric acid.
- Published
- 1980
21. Glass-bead affinity chromatography of cell attachment and spreading-promoting factors of human serum.
- Author
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Barnes DW, Mousetis L, Amos B, and Silnutzer J
- Subjects
- Animals, Cell Adhesion, Enzyme-Linked Immunosorbent Assay, Glass, HeLa Cells analysis, Humans, Laminin isolation & purification, Proteins isolation & purification, Sarcoma, Experimental analysis, Spectrophotometry, Ultraviolet, Tissue Extracts analysis, Vitronectin, gamma-Globulins analysis, Blood Proteins analysis, Chromatography, Affinity methods, Fibronectins isolation & purification, Glycoproteins isolation & purification
- Abstract
The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.
- Published
- 1984
- Full Text
- View/download PDF
22. Cytochemical evidence for the presence of phospholipids on human chromosomes.
- Author
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Gahan PB, Bartlett R, Cleland L, and Olsen K
- Subjects
- HeLa Cells analysis, HeLa Cells cytology, Histocytochemistry, Humans, Lymphocytes analysis, Lymphocytes cytology, Mitosis, Chromosomes analysis, Phospholipids analysis
- Published
- 1974
- Full Text
- View/download PDF
23. Primary structure identification of snRNAs present in highly purified snRNPs from HeLa cells.
- Author
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Sri-Widada J, Liautard JP, Assens C, and Brunel C
- Subjects
- Base Sequence, Cell Nucleus analysis, Chemical Phenomena, Chemistry, Heterogeneous-Nuclear Ribonucleoproteins, Humans, RNA, Small Nuclear, Ribonuclease T1, Ribonucleoproteins, Small Nuclear, HeLa Cells analysis, Nucleoproteins isolation & purification, RNA isolation & purification, Ribonucleoproteins isolation & purification
- Abstract
Extensive purification of snRNPs as a subset of hnRNP from HeLa cells has been previously reported (Brunel et al. (1981), Nucleic Acids Research, 9, 815). These snRNPs were shown to contain discrete RNA species comigrating in gel electrophoresis with authentic U1, U2, U4, U5 and U6 species. We now report sequence analysis data of about 50 nucleotides from the 3'-end which serve to positively establish the identity of snRNAs present in these purified snRNPs. Sequence heterogeneity was found at the 3'-end of U4 species. A minor species identical to U1 at its 3'-end but slightly shorter was identified as the U1 described by Lerner et al. (Nature (1980) 283, 220-224) through sequencing of the 5'-end. When unfixed hnRNP are centrifuged in a CsCl gradient containing 4M guanidinium chloride instead of 0.5% sarkosyl as above, a band containing only one RNA species was observed. T1 RNAse fingerprinting and sequence analysis of the oligonucleotides produced allowed identification of this RNA as U5 snRNA.
- Published
- 1981
- Full Text
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24. Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions.
- Author
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Pauli U, Chrysogelos S, Stein J, and Stein G
- Subjects
- Genes, HeLa Cells analysis, Humans, Protein Binding, Substrate Specificity, DNA, Neoplasm genetics, Deoxyribonuclease I metabolism, Nucleotide Mapping, Promoter Regions, Genetic
- Abstract
DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. We have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/-5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.
- Published
- 1988
- Full Text
- View/download PDF
25. Release of 3-methyladenine from linker and core DNA of chromatin by a purified DNA glycosylase.
- Author
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Heller EP and Goldthwait DA
- Subjects
- Adenine isolation & purification, Carbon Radioisotopes, DNA, Neoplasm isolation & purification, HeLa Cells analysis, Humans, Methylnitrosourea toxicity, Nucleosomes ultrastructure, Tritium, Adenine analogs & derivatives, Chromatin ultrastructure, DNA Glycosylases, N-Glycosyl Hydrolases metabolism
- Abstract
Oligonucleosomes were isolated from [14C]thymidine-labeled HeLa cells by digestion of the nuclei with micrococcal nuclease and were then alkylated with [3H]methylnitrosourea. Nucleosome core particles were also prepared by further digestion of the oligonucleosomes. The distribution of 3H-labeled methyl groups in the linker versus the core DNA was established by a determination of 3H:14C ratios in oligonucleosome and core DNA. The ratios in the core DNA of 145 and 165 base pair DNA fragments were 5.2 and 5.4, respectively, while the ratio in the oligonucleosomal DNA was 8.2. Assuming an equal mixture (as determined) of 145 and 165 base pair fragments of DNA in the 185 base pair repeat, the relative concentration of 3H methyl groups in the linker versus the core DNA was 4.2. Thus, 45% of the 3H methyl groups were in the linker DNA, and 55% were in the core DNA. Some shielding of the DNA was evident during alkylation. The concentrations of alkyl groups on the linker and core DNA were 67 and 12% of that found on free DNA alkylated under comparable conditions. No evidence for preferential shielding of the major or minor groove was observed. The purified 3-methyladenine DNA glycosylase I of Escherichia coli released approximately 37% of the 3-methyladenine from the linker DNA and 13% from the core DNA. The limited enzymatic removal of 3-methyladenine in vitro compared to the efficient removal in vivo suggests that conformational changes of the oligonucleosome and core structure must occur for total repair.
- Published
- 1983
26. Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.
- Author
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Kumar A, Sierakowska H, and Szer W
- Subjects
- Circular Dichroism, DNA Restriction Enzymes metabolism, Deoxyribonuclease BamHI, Heterogeneous-Nuclear Ribonucleoproteins, Humans, Isoelectric Focusing, Nucleic Acid Conformation, Peptide Mapping, Poly U metabolism, RNA Precursors metabolism, RNA Splicing, RNA, Heterogeneous Nuclear metabolism, Ribonuclease T1 metabolism, Ribonucleoproteins metabolism, Tumor Cells, Cultured analysis, HeLa Cells analysis, RNA metabolism, RNA, Heterogeneous Nuclear isolation & purification, Ribonucleoproteins isolation & purification
- Abstract
A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.
- Published
- 1987
27. Isolation and characterization of branched oligonucleotides from RNA.
- Author
-
Reilly JD, Wallace JC, Melhem RF, Kopp DW, and Edmonds M
- Subjects
- Cell Nucleus analysis, Electrophoresis, Paper methods, HeLa Cells analysis, Humans, Phosphorus Radioisotopes, Poly A isolation & purification, RNA Precursors genetics, RNA Precursors isolation & purification, RNA, Messenger, RNA, Neoplasm isolation & purification, Radioisotope Dilution Technique, Transcription, Genetic, Oligoribonucleotides isolation & purification, RNA isolation & purification
- Published
- 1989
- Full Text
- View/download PDF
28. Specific transcription of an adenoviral gene that possesses no TATA sequence homology in extracts of HeLa cells.
- Author
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Leong K and Flint SJ
- Subjects
- Base Sequence, DNA Restriction Enzymes metabolism, DNA, Viral metabolism, Electrophoresis, Polyacrylamide Gel, Humans, RNA Polymerase II metabolism, Templates, Genetic, Adenoviruses, Human genetics, Deoxyribonucleases, Type II Site-Specific, HeLa Cells analysis, Transcription, Genetic
- Abstract
Transcription of the adenovirus type 2 (Ad2) IVa2 gene, which contains no TATA-like sequence in the region immediately upstream of the IVa2 cap sites (Baker, C. C., and Ziff, E. B. (1981) J. Mol. Biol. 149, 189-221), has been examined in extracts of HeLa cells (Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3855-3859). Run-off transcripts of the predicted length of those initiated at the IVa2 cap sites were synthesized from different Ad2 DNA templates, each of which also contained the major late transcriptional control region. Mapping of the 5' ends of the RNA made from one template by a nuclease protection assay established the fidelity of initiation of IVa2 transcription in vitro. The efficiency of IVa2 expression in whole HeLa extracts was influenced quite dramatically by monovalent and divalent metal ion concentrations and the concentration of extract protein present in the reaction mixture. Under certain conditions, IVa2 run-off transcripts were made almost as efficiently as those from the Ad2 major late transcriptional control region. However, conditions promoting optimal IVa2 transcription in vitro did not favor recognition of the major late transcriptional control region, and vice versa: the synthesis of IVa2 and major late run-off transcripts responded differently to all parameters tested.
- Published
- 1984
29. Neurovirulence of the intertypic poliovirus recombinant v3/a1-25: characterization of strains isolated from the spinal cord of diseased monkeys and evaluation of the contribution of the 3' half of the genome.
- Author
-
Agol VI, Drozdov SG, Frolova MP, Grachev VP, Kolesnikova MS, Kozlov VG, Ralph NM, Romanova LI, Tolskaya EA, and Viktorova EG
- Subjects
- Animals, Chlorocebus aethiops microbiology, Genes, Viral, HeLa Cells analysis, Humans, Poliovirus genetics, Poliovirus isolation & purification, RNA, Viral analysis, Recombination, Genetic, Virulence, Poliomyelitis microbiology, Poliovirus pathogenicity, Spinal Cord microbiology
- Abstract
A tsRNA- intertypic recombinant, v3/a1-25, which has the 5' and 3' halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA +/- phenotypes, respectively, the other three strains retained the parental RNA- character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3' half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 10(3) p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3' half derived from a neurovirulent trRNA +/- type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.
- Published
- 1985
- Full Text
- View/download PDF
30. Quantitative determination of amino acids in tissue culture cells by high performance liquid chromatography.
- Author
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Kabus P and Koch G
- Subjects
- Culture Media, HeLa Cells analysis, Humans, Amino Acids analysis, Chromatography, High Pressure Liquid methods
- Published
- 1982
- Full Text
- View/download PDF
31. Purification of the major mammalian heat shock proteins.
- Author
-
Welch WJ and Feramisco JR
- Subjects
- Electrophoresis, Polyacrylamide Gel, HeLa Cells analysis, Heat-Shock Proteins, Hot Temperature, Humans, Molecular Weight, Proteins isolation & purification
- Abstract
The major mammalian heat shock or "stress" proteins (molecular masses of 90,000, 72,000, and 73,000 daltons) have been purified from stressed HeLa cells. The 90,000-dalton protein co-purified with small amounts of a 100,000-dalton protein which was identified as one of the other stress proteins in these cells. The 72,000- and 73,000-dalton proteins co-purified throughout the fractionation scheme, apparently as a mixture of monomeric forms of the two proteins. From sedimentation velocity and gel filtration analysis, it was found that the 90,000/100,000-dalton protein mixture had a Stokes radius of 69A and a s20,w value of 5.8 while the 72,000/73,000-dalton protein mixture had a Stokes radius of 42.6A and a s20,w value of 4.3. The purified proteins migrated identically in two-dimensional gel electrophoretograms with their counterparts from total cell lysates of [35S]methionine-labeled stressed HeLa cells. Peptide mapping experiments indicated that the 72,000- and 73,000-dalton proteins contained common peptides while the 90,000- and 100,000-dalton proteins appeared to be distinct. Amino acid analysis of the 90,000- and a mixture of the 72,000/73,000-dalton proteins showed that both contained relatively high amounts of Asp/Asn and Glu/Gln.
- Published
- 1982
32. The purification and quantitation of myosin from cultured cells.
- Author
-
Ostlund RE and Pastan I
- Subjects
- Adenosine Triphosphatases analysis, Animals, Cell Line, Female, HeLa Cells analysis, L Cells analysis, Methods, Mice, Myosins isolation & purification, Uterus analysis, Myosins analysis
- Abstract
Myosin has been purified from the following cultured cell lines: normal rat kidney fibroblast (NRK), HeLa-Rhino (HeLa), human choriocarcinoma, human acute lymphoblastic leukemia, rat hepatoma (HTC), monkey kidney (VERO), pigmented mouse melanoma, Y-1 rat adrenal cortex, and growth hormone-secreting GH-1. Myosin constitutes 0.5-5.4% of the protein of these cells. It was not detected in washed human erythrocytes or in two types of mouse plasmacytoma cells. Two methods for the purification of myosin from cultured cells have been employed. With Method I highly purified myosin was prepared by Sepharose 4B and DEAE-cellulose chromatography from 10(10) L-929 cells as well as from mouse uterus. Those myosins have similar molecular and subunit weights as well as ATPase activity but are immunologically distinct. Method II involving ultracentrifugation and Sepharose 4B chromatography, is suitable for the production of moderately pure myosin in good yield from as few as 5-10(7) cells (five 100-mm Petrie dishes).
- Published
- 1976
- Full Text
- View/download PDF
33. Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.
- Author
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Franke WW, Schiller DL, Hatzfeld M, and Winter S
- Subjects
- Animals, Breast Neoplasms analysis, Carcinoma, Hepatocellular analysis, Cattle, Cell Line, Cytoskeleton ultrastructure, Electrophoresis, Polyacrylamide Gel, HeLa Cells analysis, Humans, Liver Neoplasms analysis, Liver Neoplasms, Experimental analysis, Macromolecular Substances, Protein Denaturation, Rats, Solubility, Urea, Intermediate Filament Proteins isolation & purification, Keratins isolation & purification
- Abstract
Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.
- Published
- 1983
- Full Text
- View/download PDF
34. Size heterogeneity of ribosomal RNA in eukaryote evolution--1. rRNA molecular weights in species containing intact large ribosomal subunit RNA.
- Author
-
Londei P, Cammarano P, Mazzei F, and Romeo A
- Subjects
- Animals, Electrophoresis, Polyacrylamide Gel, HeLa Cells analysis, Humans, Macromolecular Substances, Molecular Weight, Plants, Species Specificity, Vertebrates, Cells analysis, Eukaryotic Cells analysis, RNA, Ribosomal analysis
- Abstract
1. The molecular weights of the two major rRNA species (L-rRNA, large ribosomal subunit RNA, and S-rRNA, small ribosomal subunit RNA) of a variety of deuterostomia, green plants and fungi have been investigated by gel electrophoresis in 99% formamide, pH 9; the overall pattern obtained under these conditions differs to some extent from that deduced by electrophoresis in neutral-salt solutions. 2. The molecular weights of the deuterostomian S-rRNA species have been conserved at a value of 0.65 X 10(6), whereas those of the L-rRNA have been kept at 1.40 X 10(6) in the lower species but have increased to 1.55 X 10(6) in birds and to 1.65 X 10(6) in mammals. 3. The molecular weights of the L-rRNA and S-rRNA components of the green plants (Dycotyledons, Monocotyledons and Gymnosperms) have been generally conserved at 1.30 X 10(6) and 0.65 X 10(6). 4. The molecular weight of the L-rRNA of the fungi has been conserved at 1.36-1.38 X 10(6), being 0.1 X 10(6) daltons heavier than that of the plants; the S-rRNA exhibits a limited degree of variability, ranging between 0.65 X 10(6) and 0.72 X 10(6).
- Published
- 1982
- Full Text
- View/download PDF
35. Relationship of mRNA from productively infected cells to the complementary strands of adenovirus type 2 DNA.
- Author
-
Tibbetts C, Pettersson U, Johansson K, and Philpson L
- Subjects
- Adenoviridae growth & development, Adenoviridae metabolism, Base Sequence, Carbon Radioisotopes, Centrifugation, Density Gradient, Chromatography, DNA, Single-Stranded isolation & purification, DNA, Viral biosynthesis, DNA, Viral isolation & purification, Humans, Hydroxyapatites, Nucleic Acid Denaturation, Nucleic Acid Hybridization, Nucleic Acid Renaturation, Serotyping, Time Factors, Transcription, Genetic, Tritium, Adenoviridae analysis, DNA, Single-Stranded analysis, DNA, Viral analysis, HeLa Cells analysis, RNA, Messenger analysis
- Abstract
The complementary strands of adenovirus type 2 (Ad2) DNA were separated by buoyant density gradient centrifugation with poly (U, G). The complementary strand DNA was shown to remain intact through the course of strand separation. The l-strand of Ad2 DNA, appearing in the less dense complex with poly (U, G) in neutral CsCl density gradients, was shown to have a buoyant density in alkaline (pH 12.5) CsCl density gradients which is 2 to 3 mg per ml greater than that of its complement (h-strand). Renaturation of purified complementary strand DNA was observed only in mixtures of h- and l-strand DNA, and then with the second-order reaction rate expected for Ad2 DNA. Hybridization of the complementary strands of Ad2 DNA with cytoplasmic mRNA isolated from infected HeLa cells was performed in liquid phase and analyzed by hydroxylapatite chromatography. Before viral DNA synthesis (6 h after infection), 13 to 18% of the h-strand and 30 to 35% of the l-strand were represented in viral mRNA. Late (18 h) after infection the mRNA represented 20 to 25% and 63 to 68% of the h- and l-strands, respectively. Most, if not all sequences present in viral mRNA before viral DNA synthesis were also present in the cytoplasm late in infection.
- Published
- 1974
- Full Text
- View/download PDF
36. Characterization of U small nuclear RNA-associated proteins.
- Author
-
Billings PB and Hoch SO
- Subjects
- Antibodies, Monoclonal, Chromatography, Affinity methods, Electrophoresis, Polyacrylamide Gel, HeLa Cells analysis, Humans, Molecular Weight, Phosphorus Radioisotopes, RNA isolation & purification, RNA, Small Nuclear, Ribonucleoproteins biosynthesis, Ribonucleoproteins, Small Nuclear, Ribonucleoproteins isolation & purification
- Abstract
Differential immunoaffinity chromatography using a combination of autoimmune antibodies allows for the rapid bulk separation of specific small nuclear ribonucleoproteins (snRNPs). Passage of a HeLa cell extract over a column constructed of human anti-Sm autoantibodies results directly in the elution of complexes containing the small nuclear RNA species, U1, U2, U4, U5, and U6, and nine major polypeptides of molecular weight 69,000, 32,000, 27,000, 26,000, 18,500, 13,000, 11,000 doublet, and less than 10,000. Passage of crude extracts through a column bearing murine monoclonal antibodies directed against the 69,000 molecular weight (U1)RNP peptide gives an enriched population of U1 snRNP particles in the retained material. When the flowthrough material from the (U1)RNP column is passed through an anti-Sm column, the retained material is enriched in U2, U4, U5 plus U6 snRNP complex. The 69,000, 32,000, and 18,500 molecular weight polypeptides are confined to the U1 fraction while the remaining proteins are recovered in both fractions. The procedure is simple and rapid, producing complexes with a high degree of resolution and in sufficient yield to provide a ready source of snRNP complexes for functional studies.
- Published
- 1984
37. The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid.
- Author
-
Bogenhagen D and Clayton DA
- Subjects
- Animals, Carbon Radioisotopes, Centrifugation, Density Gradient, Chromatography, Paper, DNA, Circular, HeLa Cells metabolism, Humans, L Cells metabolism, Mice, Molecular Weight, Mutation, Spectrophotometry, Ultraviolet, Thymidine metabolism, Thymidine Kinase metabolism, Tritium, Ultracentrifugation, DNA, Mitochondrial isolation & purification, DNA, Neoplasm isolation & purification, Genes, HeLa Cells analysis, L Cells analysis
- Published
- 1974
38. Quantitation and characterization of the microtubule associated MAP2 in porcine tissues and its isolation from porcine (PK15) and human (HeLa) cell lines.
- Author
-
Valdivia MM, Avila J, Coll J, Colaço C, and Sandoval IV
- Subjects
- Animals, Brain Chemistry, Cell Line, Clone Cells, Fluorescent Antibody Technique, HeLa Cells analysis, Humans, Microtubule-Associated Proteins, Radioimmunoassay, Swine, Tissue Distribution, Kidney analysis, Nerve Tissue Proteins isolation & purification, Proteins isolation & purification
- Published
- 1982
- Full Text
- View/download PDF
39. Plectin: a high-molecular-weight cytoskeletal polypeptide component that copurifies with intermediate filaments of the vimentin type.
- Author
-
Wiche G, Herrmann H, Leichtfried F, and Pytela R
- Subjects
- Animals, Cell Line, Electrophoresis, Polyacrylamide Gel, Glioma, HeLa Cells analysis, Humans, Microscopy, Electron, Microtubule-Associated Proteins, Molecular Weight, Plectin, Rats, Vimentin, Cytoskeleton analysis, Intermediate Filament Proteins, Muscle Proteins isolation & purification, Proteins isolation & purification
- Published
- 1982
- Full Text
- View/download PDF
40. Size distribution of poly(A)-containing messenger RNA from free polysomes of HeLa cells.
- Author
-
Murphy WI and Attardi G
- Subjects
- Centrifugation, Density Gradient, HeLa Cells analysis, Molecular Weight, Poly A analysis, Polyribosomes analysis, RNA, Messenger
- Published
- 1977
- Full Text
- View/download PDF
41. Distribution of HeLa cells polypeptides in cytoplasts and karyoplasts.
- Author
-
Bravo R, Celis A, Mosses D, and Celis JE
- Subjects
- Cell Nucleus analysis, Cytoplasm analysis, Electrophoresis, Polyacrylamide Gel, Humans, Isoelectric Focusing, Tissue Distribution, HeLa Cells analysis, Peptides metabolism
- Abstract
The polypeptide composition of cytoplasts and karyoplasts prepared form HeLa cells prelabelled with [35S]-methionine and enucleated with Cytochalasin B has been analyzed using high resolution two dimensional gel electrophoresis (IEF and NEPHGE). Of the 259 major proteins followed in this study we have identified 73 polypeptides (30 acidic(IEF) and 43 basic (NEPHGE)) that are present mainly in karyoplasts. One of these polypeptides (IEF 49) has previously been shown to be a polypeptide marker for cycling cells. A total of 59 polypeptides (27 acidic and 32 basic) were found to be present mainly in cytoplasts. Many polypeptides (109 acidic and 18 basic) including Y and beta-actin (60% in cytoplasts), beta-tubulin (60% in cytoplasts), vimentin (75% in cytoplasts) and alpha-actinin (65% in cytoplasts) were found to be present in both cellular fragments. These results could be of value in assigning the cellular distribution of potential regulatory proteins.
- Published
- 1981
- Full Text
- View/download PDF
42. Intracellular distribution of Ca2+-Mg2+ adenosine triphosphatase (ATPase) in various tissues.
- Author
-
Mughal S, Cuschieri A, and al-Bader AA
- Subjects
- Animals, Cell Nucleus analysis, Centrioles analysis, Endoplasmic Reticulum analysis, Golgi Apparatus analysis, HeLa Cells analysis, Histocytochemistry, Humans, Lymphocytes analysis, Mitochondria analysis, Rats, Rats, Inbred Strains, Ca(2+) Mg(2+)-ATPase analysis, Calcium-Transporting ATPases analysis, Spleen analysis, Thymus Gland analysis
- Abstract
The cytochemical distribution of Ca2+-Mg2+-ATPase was studied ultrastructurally, using a lead capture method at pH 8.5 and compared in various tissues. In thymic, splenic and activated peripheral blood lymphocytes and in cultured HeLa cells activity was consistently localised on the nuclear envelope, endoplasmic reticulum, Golgi apparatus, mitochondria and weakly on centrioles, but not on the plasma membrane. Intracellular activity was similarly distributed in intestinal absorptive cells where activity was particularly strong in the Golgi apparatus, and in hepatocytes where, however, activity was generally weak. Intracellular activity was lacking in renal glomerular and tubular cells and in cerebellar neurons and neuroglia. Variable activity was present on the outer surface of the plasma membrane, particularly on the brush borders of intestinal and renal tubular absorptive cells, the basolateral invaginations of distal tubules and the bile canaliculi. Mitochondrial activity, when present, was inhibited by oligomycin. The localisation at different sites may represent biochemically different ATPases including endoplasmic reticular ATPase involved in intracellular calcium regulation, oligomycin-sensitive mitochondrial ATPase, dynein-like ATPase associated with centrioles and an ectoenzyme associated with cell surface specialisations.
- Published
- 1989
43. Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site.
- Author
-
Reinberg D, Horikoshi M, and Roeder RG
- Subjects
- Adenoviridae genetics, Chromatography, DNA metabolism, Drug Resistance, HeLa Cells analysis, Heparin pharmacology, Humans, Kinetics, Peptide Initiation Factors physiology, Promoter Regions, Genetic, Transcription Factors isolation & purification, Peptide Chain Initiation, Translational, RNA Polymerase II metabolism, Transcription Factors physiology, Transcription, Genetic
- Abstract
Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.
- Published
- 1987
44. Characterization of an amino acid fucoside of normal and SV40-transformed human embryonic lung cells.
- Author
-
Morton PA, Klinger MM, and Steiner SM
- Subjects
- Amino Acids analysis, Animals, Carbohydrates analysis, Cell Transformation, Viral, Chlorocebus aethiops, Female, HeLa Cells analysis, Humans, Kidney analysis, Liver analysis, Lung embryology, Pregnancy, Simian virus 40 genetics, Fucose analysis, Glycopeptides analysis, Lung analysis
- Abstract
The incorporation of radioisotopically labeled fucose into a prominent fucosylated component, i.e., fucose-labeled amino acid fucoside 4c (FL4c), of human embryonic lung cells is markedly decreased in cell lines derived from human tumors. In the current study, we have extended the above observations by examining more closely related normal and transformed human cells, e.g., Wi38 cells and SV40-transformed Wi38 cells. We have found that the level of radioisotopically labeled fucose incorporated into FL4c of the SV40-transformed human embryonic lung cells is dramatically reduced as compared to their normal counterpart cells. Additionally, we have chemically characterized FL4c. FL4c was purified from monkey tissues using a combination of gel filtration chromatography, ion-exchange chromatography, and preparative thin-layer chromatography. Analysis of this material was accomplished by amino acid analyzer and by gas-liquid chromatography; fucose, glucosamine, and aspartic acid in molar ratios of approximately 1.0:1.0:1.0 were observed. Furthermore, alpha-L-fucosidase treatment of [3H]fucose-labeled FL4c revealed that the fucose was alpha-linked and in a terminal position. Similar treatment of [3H]glucosamine-labeled FL4c yielded a component that cochromatographed with N-acetylglucosaminylasparagine. The combined results of the structural studies are consistent with the structure of FL4c being alpha-fucosyl-N-acetylglucosaminylasparagine.
- Published
- 1982
45. Cytoplasmic and nuclear architecture in cells and tissue: form, functions, and mode of assembly.
- Author
-
Penman S, Fulton A, Capco D, Ben Ze'ev A, Wittelsberger S, and Tse CF
- Subjects
- Animals, Cell Division, Cell Transformation, Viral, Electrophoresis, Polyacrylamide Gel, HeLa Cells analysis, Humans, Microscopy, Electron, Nuclear Envelope ultrastructure, Polyribosomes analysis, Protein Biosynthesis, Cell Nucleus ultrastructure, Cytoplasm ultrastructure
- Published
- 1982
- Full Text
- View/download PDF
46. Beta-thalassemia resulting from a single nucleotide substitution in an acceptor splice site.
- Author
-
Atweh GF, Anagnou NP, Shearin J, Forget BG, and Kaufman RE
- Subjects
- Base Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Endonucleases metabolism, Gene Expression Regulation, Globins genetics, HeLa Cells analysis, Humans, Male, Mutation, Single-Strand Specific DNA and RNA Endonucleases, RNA Splicing, Thalassemia genetics
- Abstract
Beta-globin gene mutations which alter normal globin RNA splicing have confirmed the necessity of invariant nucleotides GT at donor splice sites. Functional consequences of point mutations in the invariant AG acceptor splice site have not been determined. We have isolated and characterized a beta-globin gene from a Black patient with beta-thalassemia intermedia which has an A-G transition at the usual intervening sequence 2 (IVS2) acceptor splice site. Functional analysis of transcripts produced by this mutant gene in a transient expression vector indicates that the mutation inactivates the normal acceptor splice site and results in some utilization of a cryptic splice site near position 580 of IVS2. This mutation would be expected to produce a beta-globin gene which results in no normal beta-globin mRNA.
- Published
- 1985
- Full Text
- View/download PDF
47. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I.
- Author
-
Blikstad I, Markey F, Carlsson L, Persson T, and Lindberg U
- Subjects
- Actins pharmacology, Blood Platelets analysis, Calcium pharmacology, Guanidines pharmacology, HeLa Cells analysis, Lymphocytes analysis, Macromolecular Substances, Magnesium pharmacology, Molecular Weight, Structure-Activity Relationship, Actins analysis, Deoxyribonucleases antagonists & inhibitors
- Abstract
A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.
- Published
- 1978
- Full Text
- View/download PDF
48. Use of "submarine" gel electrophoresis for running multiple two-dimensional protein gels.
- Author
-
Bhat SP and Nagineni CN
- Subjects
- HeLa Cells analysis, Humans, Isoelectric Focusing, Lens, Crystalline analysis, Electrophoresis, Polyacrylamide Gel methods, Proteins isolation & purification
- Abstract
An apparatus commonly used for the electrophoresis of submerged agarose gels was used to separate proteins in the second dimension, after isoelectric focusing in the first dimension. Multiple second-dimension gels were stacked one above the other and run horizontally, submerged in the sodium dodecyl sulfate-containing Laemmli buffer system. The reproducibility of the gels run under these conditions is remarkable and eliminates the need for individual vertical electrophoresis units for routine analysis. The units for submerged horizontal gel electrophoresis are easily made or are inexpensively available commercially.
- Published
- 1988
- Full Text
- View/download PDF
49. Tumor necrosis factor receptors in HeLa cells and their regulation by interferon-gamma.
- Author
-
Tsujimoto M and Vilcek J
- Subjects
- Cycloheximide pharmacology, Dactinomycin pharmacology, Glycoproteins genetics, Humans, RNA, Messenger biosynthesis, Receptors, Cell Surface metabolism, Receptors, Tumor Necrosis Factor, Time Factors, Tumor Necrosis Factor-alpha, Glycoproteins metabolism, HeLa Cells analysis, Interferon-gamma pharmacology, Receptors, Cell Surface analysis
- Abstract
Incubation of HeLa cell cultures with human interferon-gamma (IFN-gamma) increased the binding of radioiodinated human tumor necrosis factor (TNF) to specific cell surface receptors (TNF-R). IFN-gamma also produced a proportionate increase in receptor-mediated endocytosis of TNF. TNF-R expression was significantly increased after 6 h of exposure to IFN-gamma (100 units/ml), and it remained elevated in the continuous presence of IFN-gamma for at least 20 h. Incubation of cells with IFN-gamma in the presence of cycloheximide, followed by treatment with actinomycin D and reversal of the inhibition of protein synthesis, also resulted in increased TNF-R expression as compared to cultures subjected to the same treatments in the absence of IFN-gamma. These results suggest that IFN-gamma can directly stimulate accumulation of the mRNA for TNF-R and that TNF-R is among the cellular proteins whose synthesis is increased by IFN-gamma.
- Published
- 1986
50. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.
- Author
-
Swanson MS and Dreyfuss G
- Subjects
- HeLa Cells analysis, Heterogeneous-Nuclear Ribonucleoprotein Group C, Heterogeneous-Nuclear Ribonucleoproteins, Humans, Polyribonucleotides metabolism, RNA, Neoplasm isolation & purification, RNA, Neoplasm metabolism, Ribonucleoproteins classification, Ribonucleoproteins metabolism, Ribonucleoproteins isolation & purification
- Abstract
Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.
- Published
- 1988
- Full Text
- View/download PDF
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