Uptake of antitetanus F(ab')2 fragments into eukaryotic cells

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.     

Structural analysis of plant ribosomal 5S RNAs. Visualisation of novel tertiary interactions by cleavage of lupin and wheat 5SrRNAs with ribonuclease H

A model for the tertiary structure of plant 5S rRNA, previously proposed by our laboratory (Joachimiak, A. et al. (1990) Int. J. Biol. Macromol., in press) was tested by specific cleavage of the plant 5S rRNA in the presence of synthetic oligodeoxynucleotides. The hexanucleotides used in this study were complementary to different portions of loops C, D and E, the nucleotides of which have recently been proposed to be involved in tertiary hydrogen bonds. The results obtained strongly support the interaction of loops C and D by nucleotides C34, C35, C36, A37 and G85, G86, G87, U88, respectively. Digestion pattern of loop E (domain gamma, nucleotides 66-110) suggests a possible different arrangement of this part of the plant 5S rRNA molecule, when compared with other eukaryotes.     

Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Determination of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs by infrared spectroscopy.

The extent of base pairing in Escherichia coli and Bacillus stearothermophilus 5S RNAs was determined by infrared spectroscopy. From the infrared spectra taken at 20 degrees and 52 degrees C it is concluded that E. coli and B. stearothermophlius 5S RNAs possess a large number of base pairs (Table I). Comparison of our results with those previously published using other methods leads to the conclusion that the structures of prokaryotic 5S RNAs involve a large number of tertiary interactions, in which the base pairing is not necessarily solely of the Watson-Crick type.     

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.