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L M Roberts

Publications and source records attributed to L M Roberts.

At least 91 records · Page 5Linked to original sources
PubMed1991

The effects of N-glycosylation on the lectin activity of recombinant ricin B chain.

Soluble, biologically-active recombinant ricin B chain has been produced by expressing B chain-encoding DNA in heterologous eukaryotic or prokaryotic hosts. N-Glycosylated recombinant ricin B chain expressed in Xenopus oocytes bound to both immobilized asialofetuin and immobilized lactose. Non-glycosylated ricin B chain expressed in either E. coli or in tunicamycin-treated oocytes did not bind to immobilized lactose. However, it did bind to asialofetuin, and increasing concentrations of free lactose did not reduce this asialofetuin binding dramatically, in contrast to the effect of free lactose on the binding of either glycosylated recombinant B chain or native ricin B chain.

Amino Acid Sequence↗
PubMed1991

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.

Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.

Amino Acids↗
PubMed1991

Ribosome inactivating proteins of plants.

Many plant tissues produce single chain proteins which can enzymatically remove a specific adenine residue from ribosomal RNA. Although these proteins are potently toxic to isolated ribosomes, they are non-toxic to intact cells, being unable to gain access to their ribosomal substrate. In certain plants however, the gene for the ribosome inactivating protein has fused with a gene encoding a galactose-specific lectin. This generates heterodimeric proteins which can bind to and enter target cells, and which are among the most potent cytotoxins known.

Amino Acid Sequence↗
PubMed1990

Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence.

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

Amino Acid Sequence↗
PubMed1989

Recombinant proricin binds galactose but does not depurinate 28 S ribosomal RNA.

Preproricin transcripts microinjected into Xenopus oocytes were expressed and the product was segregated by the oocyte endoplasmic reticulum and core glycosylated. Recombinant proricin was soluble, stabilised by intramolecular disulfide bonds and biologically active in that it could bind to immobilized lactose (selectin 2) or immobilized asialofetuin. Affinity-purified proricin did not catalyse the depurination of 28 S ribosomal RNA unless it was reduced, when slight but significant activity was observed. Gel filtration of the reduced proricin fraction showed that this depurination activity was not associated with proricin. The activity was apparently due to ricin A chain released by reduction from mature ricin which was, in turn, generated from proricin, presumably via endogenous oocyte endoprotease activity.

Animals↗
PubMed1989

Expression of ricin B chain in Escherichia coli.

DNA encoding ricin B chain was fused to that encoding the E. coli OmpA signal peptide using the expression secretion vector pIN-111-ompA. When induced, E. coli cells transformed with the recombinant plasmid express ricin B chain. The recombinant product accumulates in the periplasmic space in a soluble, biologically active form.

Escherichia coli↗
PubMed1989

Ribosome inactivation by ricin A chain: a sensitive method to assess the activity of wild-type and mutant polypeptides.

When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

Amino Acid Sequence↗
PubMed1989

Trypanosoma vivax: disseminated intravascular coagulation in cattle.

Five crossbred cattle infected with Trypanosoma vivax (Likoni) by Glossina morsitans developed capillary haemorrhages at the onset of parasitaemia, followed by the presence of occult blood in faecal samples and eventually melena. Two animals required treatment to survive, on days 13 and 38 respectively. The other three animals cleared their parasitaemias without treatment. Packed cell volume (PCV) levels decreased in all animals to levels ranging from 7.5 to 17%. Relapse in a treated animal initiated marked haemorrhage and a loss of 14 PCV units during a six-day period. Thrombocytopenia was common to all animals, and thrombocytes decreased to levels of 4000/microliters of blood. All animals developed increased levels of fibrinogen and fibrin monomer. Prolonged prothrombin times were found in all animals, and activated partial thromboplastin times were also extended in the two animals with high parasitaemias.

Anemia↗
PubMed1989

Natural and acquired resistance to Trypanosoma vivax in cattle.

Zebu x European (Z x E) crossbred cattle suffered a more severe course of disease than Boran cattle when infected with Trypanosoma vivax (Likoni) by Glossina morsitans. All Z x E animals in this study required Berenil treatment while all Borans self-cured the infection without treatment. The more severe disease in Z x E animals was characterized by longer periods of patent infection and fever, more severe anaemia and greater likelihood of haemorrhage. Cattle previously infected and cured with Berenil showed resistance and self-cured challenge infections. After self-cure cattle remained immune to tsetse fly challenge with the homologous trypanosome stock for long periods. Immunity induced by infection and drug or self-cure appeared to be specific for the homologous stock, since cattle immune to T. vivax (Likoni) showed no resistance when challenged with stocks of T. vivax isolated in Lugala, Uganda or Galana, Kenya. Severe haemorrhages, most prominent in the digestive tract, were seen in some infected cattle before treatment.

Animals↗
PubMed1988

The expression of functional ricin B-chain in Saccharomyces cerevisiae.

Yeast cells transformed with plasmids containing ricin B-chain coding sequences expressed this heterologous protein. When ricin B-chain was expressed in a form which resulted in its deposition in the yeast cytosol it formed insoluble aggregates which were devoid of galactose-binding activity. In contrast, when DNA fusions were constructed, in which the B-chain coding sequence was preceded by either the preproalpha-factor leader sequence or the native preproricin signal sequence, the recombinant B-chain products were soluble and biologically active. Both the homologous yeast signal peptide and the heterologous plant signal peptide directed the expressed product into the lumen of the yeast endoplasmic reticulum. As a result, the recombinant B-chain products were processed at the N-terminus, glycosylated and folded into an active conformation, presumably stabilized by correct intrachain disulphide bond formation.

Base Sequence↗
PubMed1987

Expression of ricin A chain in Escherichia coli.

DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli. When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.

DNA↗
PubMed1987

Identification and sequencing of cDNA clones for the rodent negative acute-phase protein alpha 1-inhibitor 3.

Rat alpha 1-inhibitor 3 clones were isolated by immunological screening of a lambda gt11 cDNA library prepared from rat liver poly(A)-rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid-arrested in vitro translation. The size of the cognate poly(A)-rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of alpha 1-inhibitor 3 poly(A)-rich RNA decreases as shown by dot-blot hybridization and Northern analyses. The response of this negative acute-phase plasma protein to inflammation may therefore be considered to be at the pretranslational level. The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3'-flanking region was observed. An amino acid homology of 70% for alpha 1-inhibitor 3 with human and rodent alpha 2-macroglobulin emphasizes the evolutionary relationship of the macroglobulins.

Acute-Phase Proteins↗
PubMed1987

Plant toxins.

Many plants produce toxic proteins capable of inactivating eukaryotic ribosomes and thereby arresting protein synthesis. In certain plants the gene for a ribosome-inactivating protein has fused with a gene encoding an independent sugar-binding polypeptide to generate heterodimeric proteins which are among the most potent cytotoxins known.

Cytotoxins↗
PubMed1986

The pentafunctional FAS1 gene of yeast: its nucleotide sequence and order of the catalytic domains.

FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.

Amino Acid Sequence↗
PubMed1985

The primary sequence of Ricinus communis agglutinin. Comparison with ricin.

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.

Amino Acid Sequence↗