Induction of fusion in aggregated and nonaggregated liposomes bearing cationic detergents.

The addition of polyanionic polymers such as poly(aspartic acid) (PASP), DNA or dextran sulfate to liposomes composed of phosphatidylcholine (PC) and cholesterol (CHOL) and bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl]dimethy lbe nzylammonium hydroxide (DEBDA[OH]) resulted in liposome aggregation and fusion. Liposome-liposome fusion was studied by using fluorescently labeled liposomes and fluorescence-dequenching (DQ) methods. Addition of monoanions, such as aspartate or acetate, to liposomes bearing DEBDA[OH] caused neither their aggregation nor liposome-liposome fusion. Aggregation of liposomes bearing DEBDA[OH] by the binding pair avidin-biotin did not result in their fusion. Fusion in such aggregated liposomes was observed by the addition of chaotropic anions, such as nitrate or thiocyanate, or by PASP. A variety of other quaternary ammonium detergents behaved similarly to DEBDA[OH] in their ability to confer fusogenic properties upon PC/chol liposomes. The relevance of these findings to the mechanism of liposome-liposome fusion is discussed.

Poly(aspartic acid)-dependent fusion of liposomes bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide.

Addition of the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide (DEBDA[OH]) and the fluorescent probes N-(7-nitro-2-1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-NBD-PE and N-Rh-PE, respectively) to liposomes composed of phosphatidylcholine (PC) and cholesterol (chol) resulted in the formation of fluorescently labeled liposomes bearing DEBDA[OH]. Incubation of the anionic polymer poly(aspartic acid) (PASP) with such liposomes resulted in strong agglutination, indicating an association between the negatively charged PASP and the positively charged liposome-associated DEBDA[OH]. Addition of PASP to a mixture of fluorescently labeled and nonlabeled liposomes, both carrying DEBDA[OH], resulted in a significant increase in the extent of fluorescence, namely, fluorescence dequenching. The degree of the fluorescence dequenching was dependent upon the ratio between the nonfluorescent and the fluorescent liposomes, upon the temperature of incubation, and upon the amount of DEBDA[OH] which was associated with the liposomes. Electron microscopic observations revealed that large liposomes were formed upon incubation of liposomes bearing DEBDA[OH] with PASP. The results of the present work strongly indicate that the fluorescence dequenching observed is due to a process of PASP-induced liposome-liposome fusion.

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.

CD studies on ribonuclease A - oligonucleotides interactions.

The interaction of ApU, Aps4U, Aps4Up, ApAps4Up and Gps4U with RNase A was studied by CD difference spectroscopy. The use of 4-thiouridine (s4U) containing oligonucleotides enables to distinguish between the interaction of the different components of the ligand with the enzyme. The mode of binding of the oligonucleotides to the enzyme is described. From this mode of binding it is explained why Aps4U, for example, inhibits RNase A, while s4UpA serves as a substrate.

The preparation and properties of 4-thiouridine containing 2'-5' and 3'-5' dinucleoside monophosphates.

2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine were prepared by the thiolation of the cytosine containing compounds and purified by chromatography on a DEAE-Sephadex column. The chromatographic and optical properties of the isomers are compared.

Poly (U, s 4-U) as a synthetic messenger RNA for polyphenylalanine synthesis in a cell-free system derivem derived from rat liver.

Two different U and S4-U containing polymers with U:s4U ratio of 3:1 and 5:1 were synthesized. Their activity as messenger RNA was studied in an amino acid incorporation cell-free system from rat liver. It was shown that both copolymers can stimulate the incorporation of phenylalanine into oligophynylalamyl-tRNA.

The synthesis of poly-4-thiouridylic acid and its effect on protein synthesis in a cell free system derived from rat liver.

The synthesis of poly-4-thiouridylic acid by thiolation of polycytidylic acid is described. A quantitative thiolation was achieved without any cleavage of the phosphodiester bond. The inhibitory effect of the poly-4-thiouridylic acid on protein synthesis in a cell free system derived from rat liver was investigated.