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D Shire

Publications and source records attributed to D Shire.

At least 55 records · Page 3Linked to original sources
PubMed1992

A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin.

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

Amanitins↗
PubMed1992

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.

Amino Acid Sequence↗
PubMed1992

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Transcription of synthetic DNA containing sequences with dyad symmetry by wheat-germ RNA polymerase II. Increased rates of product release in single-step addition reactions.

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.

Base Sequence↗
PubMed1991

Human interleukin 1 beta fused to the human growth hormone signal peptide is N-glycosylated and secreted by Chinese hamster ovary cells.

A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.

Amino Acid Sequence↗
PubMed1991

Inhibition of reverse transcription by unmodified and modified antisense oligodeoxynucleotides.

We used oligodeoxynucleotides to prevent reverse transcription of beta-globin mRNA by reverse transcriptase of avian myeloblastosis virus. Unmodified oligomers hybridized to the template arrested synthesis of cDNA in a dose dependent manner. The longer the oligomer the more efficient the inhibition, 50% inhibition being achieved at 0.3 and 30 microM of a 17- or a 12-mer, respectively. The use of complementary oligonucleotides with a 3' end blocked either by a dideoxy residue or by a dodecanol group also induced inhibition of cDNA synthesis.

Animals↗
PubMed1990

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Synthetic oligodeoxynucleotides, either unmodified or linked to an intercalating agent, have been used to prevent cDNA elongation by the AMV reverse transcriptase. Oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA part bound to the antisense oligonucleotide by the RNase-H activity associated with the retroviral polymerase.

Animals↗
PubMed1990

Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli.

To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.

Amino Acid Sequence↗
PubMed1990

Effect of RNA secondary structure and modified bases on the inhibition of trypanosomatid protein synthesis in cell free extracts by antisense oligodeoxynucleotides.

Every messenger RNA from leishmanias and trypanosomes has at its 5' end a conserved region termed the mini-exon sequence which, however, varies from species to species. In a systematic study mRNAs from Trypanosoma brucei, Trypanosoma vivax, and Leishmania enriettii were translated in cell-free extracts in the presence of oligodeoxynucleotides complementary to part of the mini-exon sequence. The affinity of the same oligonucleotides for target and non-target mRNAs was determined by thermal elution of filter-bound complexes showing that the critical temperature of half-dissociation of the complexes was linearly related to log (l + x), where l is the length of the oligomer and x its G + C content. A few oligomers exhibited a lower Tc value than expected which was ascribed to the presence of modified RNA bases or to the existence of a hairpin structure in the L. enriettii mini-exon. In most cases the efficiency of translation inhibition by the oligonucleotides was clearly correlated to their affinity for the target RNA. The modified bases weakened the inhibition of protein synthesis by oligonucleotides complementary to these regions.

Animals↗
PubMed1989

Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A::growth hormone releasing factor) produced in Escherichia coli.

DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.

Base Sequence↗
PubMed1988

Potential memory and hysteretic effects in transcription.

Kinetic results of RNA-chain elongation catalysed by wheat-germ RNA polymerase II are analysed according to the concept that DNA-dependent conformational transitions of the transcription complex intervene during transcription. A model is presented, involving participation of several forms of the transcription complex with different catalytic properties, generated by the sequence and/or conformation of the DNA template and/or the experimental conditions. The available experimental data suggest that these forms are interconvertible. Examples in which hysteretic transitions might occur are outlined, such as termination of transcription and transition from abortive to productive elongation in the first steps of RNA synthesis. The slow catalytic adaptation of the transcription complex to the template sequence might be a more general phenomenon for enzyme systems acting on polynucleotide templates, in view of the recent proposal that enzyme memory effects may also have some importance in DNA replication and messenger RNA (mRNA) translation.

DNA-Directed RNA Polymerases↗
PubMed1988

Engineered rat insulin I analogue having a B16 Tyr/Asp replacement exhibits unchanged susceptibility to cleavage by insulin proteinase.

An analogue of rat insulin I was produced by oligonucleotide-directed mutagenesis of a cloned rat preproinsulin I cDNA, followed by expression of a resulting mutant gene in Escherichia coli K-12 and proteolytic cleavage of mutant proinsulin isolated from this bacterium. The Tyr-to-Asp replacement at residue B16 in the insulin analogue had been expected to diminish the rate of cleavage of the molecule by the enzyme insulin proteinase, since the bond TyrB16-LeuB17, invariant in all mammalian species, had been proposed by other authors as one of the early, major sites of proteolytic attack. In the event the substitution had no measurable effect on the rate of degradation by insulin proteinase. Thus we find no support in these experiments for the hypothesis that the site in question is of primary importance in the degradation of rat insulin I by the enzyme.

Animals↗
PubMed1988

Characterization and sequencing of normal and modified oligonucleotides by 252Cf plasma desorption mass spectrometry.

Plasma desorption (PD) mass spectra of normal deoxyribo-oligonucleotides and of neutral methylphosphonate deoxyribo-oligonucleosides are examined and discussed. Molecular ions of oligonucleotides up to nonamer have been observed for neutral species. It is also shown that PD mass spectra can be used to monitor chemical modifications of oligonucleotides, such as the covalent binding of an organic fluorescent probe, along the synthesis process.

Californium↗
PubMed1987

Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II.

A kinetic study of the effect of elongating nucleotide concentration on the reactions of abortive elongation catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template suggests that the shift from abortive to productive elongation may involve the participation of at least two nucleotides, according to a mechanism very similar to that reported for Escherichia coli RNA polymerase. Experiments performed with non-complementary nucleotides with respect to the DNA template, and with substrate derivatives, allow an analysis of the substrate specificity during these reactions. Similar experiments performed with poly[d(A-A-T)].poly[d(T-T-A)] as template provide a starting point for a better understanding of the effect of DNA sequence on the rates of abortive and productive elongation catalysed by the plant enzyme.

Adenosine Triphosphate↗
PubMed1987

Expression vector promoting the synthesis and export of the human growth-hormone-releasing factor in Escherichia coli.

We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein. A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF). The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space. Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.

Bacterial Proteins↗
PubMed1987

Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity.

A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis. The COOH-terminal domain carries the channel activity. The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated. All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium. The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed. Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity. No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity. Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance. Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels.

Amino Acid Sequence↗