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Research Interests: Biology, Tobacco, Biological Chemistry, Fluorescent Dyes and Reagents, Ion Channels, and 14 moreBiological Sciences, Cercopithecus aethiops, Humans, Animals, Rhizobium, Biological, CHEMICAL SCIENCES, HeLa cells, Protein Binding, PLANT PROTEINS, DNA binding proteins, Nuclear Import, fluorescent dyes, and Medical and Health Sciences
A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual... more
A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with ceils. By this method, ferritin and latex spheres (diameter 0.1 #m) have been "injected " into cells. The ultimate aim of a great deal of biological research is to understand the function of various types of macromolecules within the intact cell. Since many aspects of the intracellular behavior of macromolecules cannot adequately be studied in cell extracts, many laboratories have tried with varying degrees of success to introduce isolated
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Moshe Wasserman, Nehama Zakai, Abraham Loyter, and Richard G. Kulka Department of Biological Chemistry The Hebrew University of Jerusalem Jerusalem, Israel Summary Improvements in the technique of ultramicroinjec- tion of macromolecules... more
Moshe Wasserman, Nehama Zakai, Abraham Loyter, and Richard G. Kulka Department of Biological Chemistry The Hebrew University of Jerusalem Jerusalem, Israel Summary Improvements in the technique of ultramicroinjec- tion of macromolecules into animal cells are de- scribed. The method is based on the Sendai virus- induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fu- sion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more effi- cient than fusion with ghosts prepared in the pres- ence of bovine serum albumin (BSA) as in pre- vious investigations. La+++ is more efficient in promoting fusion and less toxic to cells than Mn++, which was used previously. Thus in all subsequent experiments, erythrocytes were hemolyzed in the presence of cytochrome C plus other macromole- cules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC c...
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Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled,... more
Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.
Research Interests: Genetics, Plant Biology, Heat Treatment, Plant Molecular Biology, Gene expression, and 13 moreCell Division, Plants, Glucuronidase, DNA Structure, Protoplasts, Heat Shock, Heat Shock Proteins, Transfection, Biological activity, Biochemistry and cell biology, Gene Expression Regulation, Transient Expression, and Reporter gene
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Research Interests:
A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual... more
A new method is described for the introduction of macromolecules and small particles into animal cells. The first step in this procedure is the trapping of particles in ghosts of human erythrocytes. This is achieved by the gradual hemolysis of erythrocytes in the presence of the particles to be trapped. The second step is the Sendai virus-induced fusion of the ghosts containing the particles with cells. By this method, ferritin and latex spheres (diameter 0.1 mum) have been "injected" into cells.
Research Interests: Electron Microscopy, Cell Biology, DNA viruses, Biological Sciences, Humans, and 14 moreLaTeX, Cell fusion, Plant tissue Culture Techniques, Hepatocellular Carcinoma, Microspheres, Bovine Serum Albumin, Erythrocytes, Cytoplasm, Hemolysis, Cytological Techniques, Cell Membrane, Liver neoplasms, Medical and Health Sciences, and ABO Blood-Group System
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Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled,... more
Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.
Research Interests: Genetics, Plant Biology, Heat Treatment, Plant Molecular Biology, Gene expression, and 13 moreCell Division, Plants, Glucuronidase, DNA Structure, Protoplasts, Heat Shock, Heat Shock Proteins, Transfection, Biological activity, Biochemistry and cell biology, Gene Expression Regulation, Transient Expression, and Reporter gene
Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient... more
Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.
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Research Interests:
Research Interests: Molecular Biology, RNP, HIV, Molecular, Cell line, and 15 moreHumans, Cell nucleus, Pic, Amino Acid Sequence, Virus infection, Recombinant Proteins, Enzyme Linked Immunosorbent Assay, Low molecular weight, Nuclear Import, Nuclear localization signal, Biochemistry and cell biology, NLS, Serum albumin, Molecular Sequence Data, and Nuclear Transport
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... by cytosine methylation and expression of single stranded DNA constructs Maty Hershkovitz, Yosef Gruenbaum*, Nehama Zakai and Abraham Loyter Departments ... pUC8CaMVCAT was a generous gift from Dr V. Walbot (Department of Biological... more
... by cytosine methylation and expression of single stranded DNA constructs Maty Hershkovitz, Yosef Gruenbaum*, Nehama Zakai and Abraham Loyter Departments ... pUC8CaMVCAT was a generous gift from Dr V. Walbot (Department of Biological Sciences, Stanford University). ...
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Research Interests: Time-of-flight mass spectrometry, HIV, Humans, wheat germ agglutinin (WGA), Nuclear pore complex, and 14 morePeptides, Temperature Dependence, Cell nucleus, Human immunodeficiency virus, Distilled Water, Amino Acid Profile, Amino Acid Sequence, Bovine Serum Albumin, Nuclear Import, Nuclear localization signal, Biochemistry and cell biology, Bovine serum albumin (BSA), Cell membrane permeability, and virus integration
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ABSTRACT
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Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes.... more
Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.