Nehama Zakai

Liposomes bearing a quaternary ammonium detergent as an efficient vehicle for functional transfer of TMV-RNA into plant protoplasts

Abstract Liposomes composed of phosphatidylcholine (PC) and cholesterol, and bearing the hydroxyl form of the quaternary ammonium detergent diisobutylcresoxyethoxyethyldimethylbenzylammonium (DEBDA [OH−]),were able to mediate functional transfer of tobacco mosaic virus RNA (TWV RNA) into tobacco and petunia protoplasts. Functional transfer of TMV RNA was revealed by the appearance of specific capsid polypeptides within 48 h after transfection. Transfer of TMV RNA with the aid of liposomes bearing the quaternary ammonium detergent was achieved using two alternative methods. TMV RNA was enclosed within large unilamellar liposomes composed of phosphatidylcholine and cholesterol, and bearing positively charged detergent. Incubation of such liposomes loaded with TMV RNA with plant protolasts resulted in transfer of the enclosed RNA into the recipient protoplasts. In addition, functional transfer was also achieved by a complex formed between liposomes bearing the detergent and externally added (non-enclosed) TMV RNA. Maximum transfection was obtained with a complex composed of lipids, a quaternary ammonium detergent and TMV RNA, at a ratio of 1:0.5:0.035 (w/w). Using such a complex, under optimal conditions, about 30% of the recipient protoplasts were transfected with TMV RNA. Liposomes lacking the quaternary ammonium detergent practically failed to transfer TMV RNA into tobacco or petunia protoplasts.

The Plant VirE2 Interacting Protein 1. A Molecular Link between the Agrobacterium T-Complex and the Host Cell Chromatin?

The microbe Agrobacterium tumefaciens is harmful to plants and useful to scientists for one and the same reason: It transfers DNA into plant genomes. Found in soil worldwide, Agrobacterium causes disease in plants by transferring its own DNA into plant cells. But in the laboratory, the ability to

Fusion of human erythrocyte ghosts promoted by the combined action of calcium and phosphate ions.

CALCIUM ions are important in almost all membrane fusion systems1–2. Murayama and Okada3 showed that Sendai virus-induced fusion of Ehrlich ascites cells required Ca2+. When Ehrlich ascites cells were treated with Sendai virus in the presence of EDTA, cells agglutinated and lysed but did not fuse3,4. Similarly, Ca2+ was required for the fusion of erythrocytes stimulated by chemicals such as lysolecithin5 or glyceryl monooleate6. Ca2+ plus the ionosphere A23187 (ref. 7) or Ca2+ at high pH (ref. 8) promoted the fusion of chicken erythrocytes. Fusion of phosphatidylserine-rich liposomes was absolutely dependent on the presence of Ca2+9. We describe here the fusion of human erythrocyte ghosts promoted by the combined action of Ca2+ and phosphate buffer. Human erythrocyte ghosts, because they lack cytoplasm and are easily filled with small or large molecules10,11 are useful for the investigation of membrane fusion.

A quantitative study of ultramicroinjection of macromolecules into animal cells

Improvements in the technique of ultramicroinjection of macromolecules into animal cells are described. The method is based on the Sendai virus-induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fusion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more efficient than fusion with ghosts prepared in the presence of bovine serum albumin (BSA) as in previous investigations. La+++ is more fficient 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 macromolecules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC cells which fused with ghosts reached 80% in many experiments. Ghosts containing 125I-BSA were used to measure the number of BSA molecules injected into HTC cells. About 10(6) BSA molecules were injected per fused cell. The overall efficiency of injection was low (about 0.02% of the starting material).

A peptide derived from the N-terminal region of HIV-1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr.

Viral protein r (Vpr), a HIV‐1 auxiliary protein which mediates nuclear import of the viral preintegration complex (PIC), contains two regions, N‐ and C‐terminal, which have been proposed to function as a nuclear localization signal (NLS). We have synthesized peptides corresponding to both regions (designated as VprN and VprC), conjugated them to bovine serum albumin (BSA), and tested their ability to mediate nuclear import in permeabilized cells. Only VprN, and not VprC, functioned as an active NLS and promoted translocation of the conjugate into nuclei. Nuclear import of the conjugate was found to be energy and temperature dependent and was inhibited by wheat germ agglutinin (WGA). However, it did not require the addition of cytosolic factors and was not inhibited by the prototypic SV40 large T‐antigen NLS peptide. Our results show that Vpr harbours a non‐conventional negatively charged NLS and therefore suggest that Vpr may use a distinct nuclear import pathway.

Transient expression of the plasmid pCaMVCAT in plant protoplasts following transformation with polyethyleneglycol

Petunia and carrot protoplasts have been transformed with the plasmid pCaMVCAT by the use of polyethyleneglycol (PEG) as a facilitator. Transformation was revealed by the appearance of the chloramphenicol-acetyl transferase (CAT) enzyme within the transformed cells. Maximal activity of the CAT enzyme was detected within 15 h following transformation, while after 60 h, its activity was significantly reduced, indicating transient expression of the CAT gene. The efficiency of transformation was highly dependent on the presence of CaCl2 in the transformation system, was stimulated by non-functional carrier DNA and was independent on the molecular weight (MW) of PEG used.

A possible involvement of virus-associated protease in the fusion of sendai virus envelopes with human erythrocytes

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.

Membrane-bound antiviral antibodies as receptors for Sendai virions in receptor-depleted erythrocytes

Anti-Sendai virus antibodies were covalently coupled to neuraminidase-treated human erythrocytes by the use of the bifunctional crosslinking reagents, N-succinimidyl-3-(2-pyridyldithio)propionate or succinimidyl-4-(p-maleimidophenyl)butyrate. Neuraminidase-treated erythrocytes bearing antibodies were able to bind Sendai virus particles, while treated erythrocytes lacking the antibodies failed to do so. Virus particles attached to erythrocyte membranes via the antibodies were able to cause hemolysis (virus-cell fusion) and promoted cell-cell fusion. Similar results were obtained when the antibodies were coupled to cat erythrocytes which lack receptors for Sendai virus particles. Reconstituted Sendai virus envelopes, similar to intact virus particles, were able to hemolyze and to induce fusion of neuraminidase-treated antibody-bearing erythrocytes. However, reconstituted envelopes containing inactive HN (hemagglutinin-neuraminidase) but active F (fusion) glycoproteins, despite attachment to antibody-bearing erythrocytes, failed to hemolyze or to induce cell-to-cell fusion. Fusion could be restored by insertion of an active HN glycoprotein into the membranes of the reconstituted envelopes. These results suggest that the HN glycoprotein, besides being the viral attachment protein, also participates in the membrane fusion process.

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor.

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.

Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts

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.