Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on multilipid systems.

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.

A modified approach to identification of the sickle cell anemia mutation by means of allele-specific polymerase chain reaction.

The allele-specific PCR approach has been modified by introducing a second mismatch at the 3'-penultimate link of the primer and used to identify the sickle cell anemia mutation (A-->T transversion in the sixth codon of the human beta-globin gene causing Glu-->Val substitution in the protein), thus obviating the problem of an interpretationally ambiguous 3'-terminal mismatch including T residue.

Method of artificial DNA splicing by directed ligation (SDL).

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary.

Two-dimensional NMR study of the conformation of (34-65)bacterioopsin polypeptide in SDS micelles.

The conformation of the synthetic 32-residue polypeptide, an analog of the membrane spanning segment B (residues 34-65) of Halobacterium halobium bacterioopsin, incorporated into perdeuterated sodium dodecyl sulfate micelles in the presence of trifluoroethanol was investigated by 1H NMR spectroscopy. The spectrum resonances were assigned by means of phase-sensitive DQF-COSY, TOCSY and NOESY techniques. Interproton nuclear Overhauser effects and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra.

Influence of ganglioside GM3 and its breakdown products on lymphoblastic transformation and T-suppressor activity.

The influence of ganglioside GM3 and some of its breakdown products on phytohemagglutinin-induced blast transformation of human lymphocytes and concanavalin-A-induced T-suppressor activity was studied. The structures of two major hydrolysis products of GM3 were established by negative-ion fast-atom-bombardment mass spectrometry as neuraminyllactosylsphingosine (NeuLacSph) and neuraminyllactosylceramide (NeuLacCer). Both substances were shown to be potent inhibitors of mitogen-induced lymphoblastic transformation whereas their acetylation products NeuAcLacSphAc and GM3 did not affect the proliferative response of lymphocytes to phytohemagglutinin.

Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases.

A method for preparative in vitro mRNA synthesis is presented. The method makes it possible to generate several hundred RNA copies per molecule of linearized DNA template. The protocol is applicable to the synthesis of RNAs of differing lengths (from 200 to 3000 bases), and both SP6 and T7 RNA polymerases can be used.

Linkage mapping of the human CSF2 and IL3 genes.

Interleukin 3 (encoded by the IL3 gene) and granulocyte-macrophage colony-stimulating factor (encoded by the CSF2 gene) are small secreted polypeptides that bind to specific cell surface receptors and regulate the growth, gene expression, and differentiation of many of the hematopoietic cell lineages, particularly nonlymphoid cells. The IL3 and CSF2 genes have been cloned and mapped to human chromosome bands 5q23-31. Only 10 kilobases of DNA separates the two genes, suggesting that they have a common origin and/or regulation.

Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences.

Studies on the N-terminal sequences of lectins isolated from the seeds of Butea frondosa.

Two lectin fractions (FI and FII) were obtained from seeds of Butea frondosa by affinity chromatography on a sorbent of macroporous glass coupled to the disaccharide alpha-D-GalNAc-(1----3)-beta-D-Gal. Both of these fractions, although different in their sugar specificity, were found on SDS-PAGE to consist of two polypeptide chains of 33 kDa and 35 kDa. In the native state the subunits associated to form a 250 kDa complex, possibly comprising four molecules of the 33 kDa polypeptide and four molecules of the 35 kDa polypeptide.

Synthesis and modification of genes through artificial splicing by directed ligation (ASDL).

An approach to the directed genetic recombination in vitro mediated by synthetic oligodeoxynucleotides and polymerase chain reaction (PCR) is devised, which allows the joining, in a predetermined chemical-enzymatic way, of a series of DNA segments to give a precisely spliced polynucleotide sequence (Artificial Splicing by Directed Ligation, ASDL). The approach can thus lead to the totally processed eukaryotic genes using genomic DNA, with no mRNA needed. This approach has been used for the synthesis of artificial genes of interleukin-1 alpha and, in combination with PCR on the mRNA-cDNA duplex as template, of interleukin-1 receptor antagonist and their analogues, as well as for the modified genes.

Construction and properties of fusion proteins between human interferon-gamma and human tumour necrosis factors alpha and beta.

A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes. In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence. A series of mutants with deletions in the interferon part of the fusion proteins were also produced.

Functional expression in vitro of bovine visual rhodopsin.

Expression of functionally active bovine visual rhodopsin was achieved by sequential transcription and translation in vitro of rhodopsin gene cDNA with co-translational insertion of the protein into phosphatidylcholine liposomes. The recombinant rhodopsin has functional, spectral and immunochemical properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins, Cys316----Ser and Asp330----Asn, Asp331----Asn, were produced by oligonucleotide-directed mutagenesis.

Antigenic determinants synthesized in a library of randomly cloned fragments of the HIV-1 genome.

A DNA expression library of randomly selected fragments of the HIV-1 genome was constructed and used to search for antigenic determinants. A large segment of the HIV-1 provirus was sonicated, and 150-250 bp DNA fragments were cloned in a system of expression vectors developed to obtain high yields of recombinant proteins in Escherichia coli. The expressed library was immunoscreened with sera of AIDS patients.

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity.

The role of glycosphingolipids in natural immunity. Gangliosides modulate the cytotoxicity of natural killer cells.

Incubation of gangliosides with natural killer (NK) cells from various sources was found to inhibit NK activity in vitro whereas incubation of the same gangliosides with human or mouse lymphoma cells prior to their exposure to NK effectors resulted in a sharp increase in the NK sensitivity of the tumor cells. These effects depended on the oligosaccharide structure of the gangliosides and on the origin of the NK effector cells. The lysis of YAC cells by mouse splenocytes or of MOLT-4 cells by NK cells isolated from the peripheral blood of Syrian hamsters or humans was inhibited most strongly by pre-incubation of the effector cells with gangliosides GM3 and GD3 which are known to be elevated in the serum of tumor-bearing hosts.

Effective method for obtaining long nucleotide chains on partially complementary templates. Processed bovine gamma-interferon gene obtained from human gamma-interferon gene.

The method of obtaining the bovine gamma-interferon gene by means of simultaneous multidirected mutagenesis of the human gamma-interferon gene is presented. The first strand of the bovine gamma-interferon gene was obtained by ligation of synthetic oligonucleotides, using the cDNA of human gamma-interferon, cloned in the single-stranded phage M13mp19 as a template. The second strand was synthesized using a large fragment of E.

Variations in genome fragments coding for RNA polymerase in human and simian hepatitis A viruses.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e.