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Biomedical subjects

D J Ecker

Publications and source records attributed to D J Ecker.

At least 19 recordsLinked to original sources
PubMed1992

Implication of RNA structure on antisense oligonucleotide hybridization kinetics.

A 47-nucleotide transcript of the activated Ha-ras gene was prepared and determined, by enzymatic structure mapping, to form a stable hairpin structure. Six antisense decaribonucleotides were designed, and association constants (Ka) for the hairpin- and length-matched complements were measured. Two of the antisense oligonucleotides targeted to the loop had nearly equal affinity for the transcript compared to the complement. The others, including one oligonucleotide complementary to the 3' side of the single-stranded loop, bound 10(5)-10(6)-fold less tightly to the transcript than to the short complement. We propose the difference in affinity is due to the target structure, both the secondary structure of the stem and the structure in the loop. Measurement of the bimolecular association rate constant, k1, and the dissociation rate constant, k-1, for these oligonucleotides indicates the observed relationship between affinity and structure is primarily due to k1.

Base Sequence↗
PubMed1992

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.

Amino Acid Sequence↗
PubMed1992

Pseudo--half-knot formation with RNA.

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.

Base Sequence↗
PubMed1992

Enhancement of ribosomal frameshifting by oligonucleotides targeted to the HIV gag-pol region.

The pol gene of all retroviruses is expressed as a gag-pol fusion protein which is proteolytically processed to produce all viral enzymes. In the human immunodeficiency virus (HIV), the gag and pol genes overlap by 241 nucleotides with pol in the -1 phase with respect to gag. The gag-pol fusion is produced via a -1 ribosomal frameshifting event that brings the overlapping, out-of-phase gag and pol genes into translational phase. Frameshifting occurs at a so called 'shift site' 8-10 nucleotides upstream of a hairpin loop which may play a role in the regulation of frameshifting. We have fused this region of HIV-1 to the 5' end of the firefly luciferase reporter gene in order to quantitatively measure ribosomal frameshifting both in cells and by in vitro translation. A series of 2'-O-methyl oligonucleotides was designed to specifically bind the sequences which flank the gag-pol hairpin. Ribosomal frameshifting is enhanced up to 6 fold by those oligonucleotides which bind the area just 3 to the stem. Oligonucleotides which bind 5' to the stem have no effect on frameshift efficiency. In addition, we have constructed a series of fusion genes which mimic the effect of the bound oligonucleotides with intramolecular hairpins. The results suggest that increasing RNA secondary structure downstream of the shift site increases the frequency of ribosomal frameshifting, and that this effect can be mimicked by antisense oligonucleotides.

Amino Acid Sequence↗
PubMed1991

Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.

Antiviral Agents↗
PubMed1991

Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element.

All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat.

Alkaline Phosphatase↗
PubMed1990

A human ubiquitin carboxyl extension protein functions in yeast.

The ability of the human ubiquitin carboxyl extension protein (HUBCEP80) to functionally replace its yeast homolog was determined in a ubi3 mutant of Saccharomyces cerevisiae. Expression of HUBCEP80 in ubi3 mutants resulted in processing of the fusion protein to produce free ubiquitin and extension protein, the latter of which localized specifically with the 40 S ribosomal subunit. Furthermore, expression of the human fusion protein completely alleviated the phenotypic deficiencies found in ubi3 mutants, including slow growth, abnormal ribosomal RNA processing, and correspondingly low levels of 40 S ribosomal subunits. Finally, expression of the extension protein alone was much less efficient in complementing the ubi3 mutant phenotype as compared with expression of the normal ubiquitin-fused extension protein. In the latter case, cells were found to contain at least 5-fold more extension protein, suggesting that ubiquitin either increased translational efficiency of the HUBCEP80 transcript or increased the stability of the processed extension protein.

Chromosome Deletion↗
PubMed1989

Multiple (alpha-NH-ubiquitin)protein endoproteases in cells.

Ubiquitin is encoded as a variable, spacerless repeat of the gene terminating with an additional amino acid or as a gene coding for a single ubiquitin with a carboxyl-terminal extension of 52 to 80 amino acids. We report the identification and partial purification of enzymes that specifically hydrolyze the peptide bond between ubiquitin-ubiquitin conjugate (Ub-Ubase) or ubiquitin fusion proteins (Ub-Xase). The Ub-Ubase was separated from the Ub-Xase by dye-ligand-Sepharose chromatography. The Ub-Xase was purified further by affinity chromatography on ubiquitin-Sepharose. The fidelity of the endoprotease reaction was assessed by measuring the ability of the released ubiquitin to be activated by ubiquitin-activating enzyme (E1) which requires intact ubiquitin and by sequence analysis of the released carboxyl extension protein with 52 amino acids after endoproteolysis of human ubiquitin with 52-amino acid carboxyl extension. The failure of both Ub-Ubase and Ub-Xase to cleave a mutant ubiquitin-Gly-76----Ala-metallothionein showed that the endoproteases distinguish Gly-X from an Ala-X peptide bonds.

Animals↗
PubMed1989

Genetic regulation of beta 2-adrenergic receptors in 3T3-L1 fibroblasts.

The beta 2-adrenergic receptor from mouse 3T3-L1 cells is up-regulated through genetic mechanisms by glucocorticoids and butyrate. To study the genetic regulation of these receptors, we sequenced a 5 kb region of genomic DNA from 3T3-L1 cells, containing the beta-adrenergic receptor gene and approx. 1.5 kb of both 5' and 3' flanking sequences. The sequence contained one copy of an 8 bp consensus sequence which can confer phorbol ester-responsiveness to genes. Phorbol esters attenuated the up-regulation of beta 2-adrenergic receptors by glucocorticoids but not by butyrate. This effect was probably due to a phorbol ester-induced decrease in glucocorticoid receptor number. Using methylation-sensitive restriction enzymes, we examined the methylation of a CG-rich region occurring 5' to the gene and did not detect any changes in methylation of this region upon dexamethasone or butyrate treatment. A total of 16 putative glucocorticoid response elements were found which may mediate the glucocorticoid-induced increase in beta 2-adrenergic receptors. A comparison of the regulatory sequences of the two beta-adrenergic receptor subtypes from human and mouse confirms the observed physiological controls of receptor subtype expression and offers an explanation as to why the subtypes differ in genetic regulation.

Animals↗
PubMed1989

Increasing gene expression in yeast by fusion to ubiquitin.

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

DNA Mutational Analysis↗
PubMed1989

Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins.

In order to study 1) the mechanisms responsible for generating free ubiquitin monomer and 2) the function of ubiquitin carboxyl extension proteins in eukaryotes, we have developed a system for expression of human ubiquitin carboxyl extension proteins in prokaryotic and eukaryotic hosts. When expressed in Saccharomyces cerevisiae, the intact ubiquitin carboxyl extension proteins were rapidly processed to free ubiquitin monomer and extension protein. Furthermore, expression in this host conferred a slow growth phenotype mediated by the extension protein. Expression in Escherichia coli did not result in processing of the fusion proteins. However, when the expressed fusion proteins were purified from E. coli and incubated with a rabbit reticulocyte extract, the proteins were rapidly processed to free ubiquitin monomer and extension protein. These results show that human ubiquitin carboxyl extension proteins are processed to ubiquitin and extension protein when expressed in eukaryotic but not prokaryotic cells and that pre- and co-translational events are not necessary for their processing. Establishment of this system will allow for large scale purification of these proteins which will aid future studies on the function and structure of ubiquitin carboxyl extension proteins, as well as the mechanisms responsible for their processing.

Amino Acid Sequence↗
PubMed1989

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.

Computer Simulation↗
PubMed1989

Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.

Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes. An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins. Fusion of ubiquitin to yeast metallothionein or to the alpha subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein. A ubiquitin-N alpha-protein hydrolase has been partially purified from rabbit reticulocytes; this enzyme faithfully cleaves the junction peptide bound between the C-terminal Gly-76 of ubiquitin and the fusion protein. The increased yield of cloned gene products is very likely due to increased stability and/or more efficient translation of the fusion proteins. Possible mechanisms for the augmentation of ubiquitin fusion-protein expression in prokaryotes and eukaryotes are discussed.

Cloning, Molecular↗
PubMed1988

Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions.

We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein. We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein. The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein. Site-directed mutants of ubiquitin-metallothionein expressed in yeast were used to study the specificity of the (alpha-NH2-ubiquitin) protein endopeptidases. The data suggest that amino-terminal ubiquitination is not a signal for the proteolysis of yeast metallothionein in yeast. We have also discovered that expression of selected ubiquitin mutants blocked the growth of yeast. The data suggest that in addition to its function as a proteolytic signal, ubiquitination of proteins plays multiple roles in the cell.

Copper↗
PubMed1988

Ubiquitin carboxyl-terminal peptides. Substrates for ubiquitin activating enzyme.

The carboxyl terminus of ubiquitin is activated in the presence of ATP to enter the ubiquitin cycle in cells. Peptides corresponding to the COOH-terminal region of ubiquitin were synthesized to investigate their effects on the ATP/ubiquitin-dependent proteolytic pathway. Their activities in the PPi exchange assay with ubiquitin activating enzyme (E1) were proportional to their length. The hexapeptide Ac-Leu-Arg-Leu-Arg-Gly-Gly reacted with ATP to form an enzyme-adenylate-hexapeptide complex and at high concentrations was 20-25% as active as human ubiquitin in the PPi exchange assay with E1. However, the hexapeptide was not transferred to the sulfhydryl "thiol" site on E1. In addition, the COOH-terminal peptides did not support the degradation of 125I-bovine serum albumin in the reticulocyte lysate system. A nonhomologous peptide of equivalent length was inactive in all assays. Thus, synthetic COOH-terminal peptide(s) of ubiquitin can partially substitute for ubiquitin in its reactions with E1 but do not support subsequent steps of the energy-dependent proteolytic pathway. These results show that it may be possible to design small molecules that either serve as substrates or inhibitors for other specific steps in ubiquitin-dependent pathways.

Adenosine Triphosphate↗