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Biomedical subjects

M E Hogan

Publications and source records attributed to M E Hogan.

At least 55 records · Page 3Linked to original sources
PubMed1994

Inhibition of T7 and T3 RNA polymerase directed transcription elongation in vitro.

A class of oligonucleotides which binds to naturally-occurring duplex DNA sites at physiologic pH to form triple helical structures was used as transcription attenuators in an in vitro transcription assay. Oligonucleotides were designed to form triple helices with a purine-rich, double-stranded target by binding in the major groove in an orientation anti-parallel to the most purine-rich strand of the target. A 45 base-pair purine-rich region located within the gag gene of Friend Murine Leukemia Virus (FMLV) was used as the duplex target. The target DNA was inserted by molecular cloning downstream of either the bacterial T7- or T3 promoter. The sequence-specific interaction of the triple helix-forming oligonucleotide (TFO) with the FMLV target was confirmed by DNAse I footprint analysis. The affinity of the TFO, as measured by the equilibrium dissociation constant of the TFO for the duplex, was determined by band shift analysis. When a TFO was allowed to form a triple helix with the target duplex in well-defined buffer conditions before the transcription reaction, truncated transcripts of a predicted size were observed. Attenuation of transcription was observed only when buffer conditions favorable to triple helix formation were used. In addition, oligonucleotides containing a high percentage of guanosine residues were able to inhibit mRNA production of the bacterial T7 polymerase by a mechanism independent of transcription attenuation. The ability of an oligonucleotide-directed triple helical structure to slow down, or even completely stop, RNA chain elongation may expand the utility of triple helix technology in the area of gene regulation.

Bacteriophage T3↗
PubMed1993

Thermodynamics and premelting conformational changes of phased (dA)5 tracts.

Using synthetic 45 bp long DNAs of known sequences, we have studied (i) the unusual structure that the phased A-tracts have at temperatures below 37 degrees C, (ii) the thermodynamics of the loss of that aberrant structure well below the duplex melting temperature, and (iii) the conformational changes that occur with temperature. Using temperature-dependent circular dichroism, we detect a low-temperature structural transition in a 45-mer duplex with four segments of phased (dA)5 tracks separated by five segments of five randomized G-C pairs, but none in the corresponding isomeric random sequence 45-mer duplex. Differential scanning calorimetry measurements reveal the enthalpy of this preglobal melting transition to be 3.5 kcal/mol.AT pair or 4.4 kcal/mol.AA step. The integrated enthalpy change for this helix-to-helix intramolecular event only is about 16% of the global duplex-to-single-strands melting enthalpy and is relatively broad compared to the global melting event (about 30 vs 15 degrees C for the full width at half maxima). Electric birefringence decay measurements show that the phased 45 bp duplex has a rotational time constant of 100 ns at 5 degrees C which increases to 220 ns above 40 degrees C. Simple modeling of the dynamics within the junction model for bending yields that the bend per A-tract is 42 degrees at 5 degrees C, decreasing to 0 degrees above 40 degrees C. We suggest that this sequence-dependent structure which "melts" at physiological temperatures may be relevant to DNA topology and function.

Adenine Nucleotides↗
PubMed1993

Triple helix formation at distant sites: hybrid oligonucleotides containing a polymeric linker.

An oligonucleotide hybrid is described which possesses two triple helix forming oligonucleotides which have been connected by a flexible polymeric linker chain. As a prototype, binding of this class of oligonucleotide to duplex DNA has been studied using a segment of the HSV-1 D-glycoprotein promoter, which possesses a pair of 12bp target sites for stable triple helix formation, separated by a duplex spacer region which is one helical turn long. Band shift and footprinting analysis show that such hybrids can bind to both 12bp elements simultaneously, if flexible linkers are included which are longer than 20-25 rotatable bonds. Molecular modeling confirms that a flexible polymeric linker as short as 22 rotatable bonds is enough to link the two distant segments of triple helix, providing that the linker element travels a path which is external to the helix grooves and parallel to the long helix axis.

Base Sequence↗
PubMed1993

In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide.

Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential.

Animals↗
PubMed1993

Synthesis of cholesteryl supports and phosphoramidites containing a novel peptidyl linker for automated synthesis of triple-helix forming oligonucleotides (TFOs).

Preparation of G-rich TFOs containing cholesterol at the end presents challenging problems due to low yield in synthesis and loss in purification due to aggregation. These compounds also have low solubility which can result in variability in their uptake properties and cellular efficacy. To overcome these problems we have designed and synthesized cholesteryl -CPG (6) and -TentaGel (7) supports and phosphoramidite (8) containing a triglycyl linker.

Cholesterol↗
PubMed1992

The vacuum UV CD spectra of G.G.C triplexes.

Vacuum UV circular dichroism (CD) spectra were measured down to 175 nm for d(C)10, d(G)10, the d(G)10.d(C)10 duplex, and the d(G)10.d(G)10.d(C)10 triplex. A CD difference spectrum was calculated for d(G)10.d(C)10 giving the change in CD induced by forming the duplex from d(G)10 and d(C)10. The d(G)10.d(G)10.d(C)10 CD difference spectrum gave the CD induced by triplex formation from binding of d(G)10 to the d(G)10.d(C)10 duplex. In the near-UV, the d(G)10.d(C)10 and d(G)10.d(G)10.d(C)10 difference spectra resembled the difference spectrum for poly[r(G).r(C)] (Biopolymers 29, 325-333). This similarity may be an indication of similar purine base stacking. The d(G)10.d(G)10.d(C)10 vacuum UV difference spectrum had a negative band at 195 nm and a positive band at 180 nm, making it similar to difference spectra for homopolymer triplexes containing T.A.T and U.A.U triplets (Nucl. Acids Res. 19, 2275-2280). The appearance of these bands in difference spectra should be good indicators of triplex formation. The complementary oligonucleotides c-mycI d(CCCCACCCTCCC) and c-mycII d(GGGAGGGTGGGG) are part of the regulatory sequences of the human c-myc gene. G.G.C rich triplexes formed by binding c-mycII or c-mycIII d(GGGGTGGGTGGG) to the c-mycI.c-mycII duplex had CD difference spectra similar to that of d(G)10.d(G)10.d(C)10 in both the vacuum UV and near UV regions, indicating similar triplet structures.

Base Sequence↗
PubMed1992

Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U937). Oligonucleotides HIV31 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-3') and HIV38 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-3') were designed to interact with the transcription initiation site (-16 to + 13) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a 3' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+3-3') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The 3' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV31 or HIV38 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U937, was treated with 10 microM HIV38. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to alpha-interferon induction resulting from oligonucleotide treatment. Both HIV31 and HIV38 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.

Base Sequence↗
PubMed1992

In vivo stability and kinetics of absorption and disposition of 3' phosphopropyl amine oligonucleotides.

Development of oligonucleotide derivatives as therapeutic agents requires an understanding of their pharmacokinetic behavior. The in vivo disposition and stability of a prototype of such compounds are reported here. The compound studied, a relatively G-rich 38 base 3' phosphopropyl amine oligonucleotide (TFO-1), was cleared from the circulation with a half-life of approximately 10 minutes, displaying distribution kinetics consistent with a two compartment model. TFO-1 was also readily absorbed into circulation from the peritoneal cavity. All tissues examined except brain accumulated the compound reaching concentrations calculated to be in the micromolar range. TFO-1 was found to be stable in circulation and in tissues in that a large fraction of intact material was detected 8 hours after injection, as assessed by gel electrophoresis. Approximately 20-30% of the injected dose was excreted in the urine over an 8 hour period. These results suggest that G-rich oligonucleotides, minimally modified at the 3' end, are relatively stable in vivo and have distribution kinetics favorable to use as therapeutic agents.

Absorption↗
PubMed1992

Human therapeutics based on triple helix technology.

The application of triple helix technology to rational drug discovery is rapidly leading to the development of a new class of drugs, initially applicable for the effective and specific treatment of viral infections and, eventually, perhaps, cancer and immunological disorders. Issues such as stability, delivery, specificity, in vitro efficacy and acute toxicity, and the cost of synthesis are already being addressed: preliminary studies are yielding promising results. Further chemical modification of the oligonucleotides will probably be necessary to enhance affinity and efficacy, and comprehensive studies on the pharmacokinetics, toxicity, mutagenicity and in vivo efficacy of these compounds are still required. These, as well as scale-up synthesis and pharmaceutical formulation issues, are the focus of the R&D programs of a number of pharmaceutical laboratories.

Base Sequence↗
PubMed1991

Binding of triple helix forming oligonucleotides to sites in gene promoters.

A class of triplex-forming oligodeoxyribonucleotides (TFOs) is described that can bind to naturally occurring sites in duplex DNA at physiological pH in the presence of magnesium. The data are consistent with a structure in which the TFO binds in the major groove of double-stranded DNA to form a three-stranded complex that is superficially similar to previously described triplexes. The distinguishing features of this class of triplex are that TFO binding apparently involves the formation of hydrogen-bonded G.GC and T.AT triplets and the TFO is bound antiparallel with respect to the more purine-rich strand of the underlying duplex. Triplex formation is described for targets in the promoter regions of three different genes: the human c-myc and epidermal growth factor receptor genes and the mouse insulin receptor gene. All three sites are relatively GC rich and have a high percentage of purine residues on one strand. DNase I footprinting shows that individual TFOs bind selectively to their target sites at pH 7.4-7.8 in the presence of millimolar concentrations of magnesium. Electrophoretic analysis of triplex formation indicates that specific TFOs bind to their target sites with apparent dissociation constants in the 10(-7)-10(-9) M range. Strand orientation of the bound TFOs was confirmed by attaching eosin or an iron-chelating group to one end of the TFO and monitoring the pattern of damage to the bound duplex DNA. Possible hydrogen-bonding patterns and triplex structures are discussed.

Animals↗
PubMed1991

Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.

A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.

Base Sequence↗
PubMed1991

Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes.

The promoter region of the IL2R alpha gene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors which upregulate transcription following T lymphocyte activation. A 3' exonuclease resistant oligonucleotide (3'A-IL28p, terminated by a free amine group at its 3' end) was designed to bind to the IL2R alpha promoter region from -273 to -246, forming a collinear triplex spanning the kappa B enhancer (-266 to -256) as well as most of the serum response element (CArG box, -251 to -244). The binding site specificity of this oligonucleotide was demonstrated in electrophoretic mobility shift assays and by inhibition of restriction endonuclease (HinfI) cleavage within the segment of the target DNA predicted to form a triplex with the oligonucleotide. Intact normal lymphocytes, preincubated for 2h with 3'A-IL28p, accumulated less IL2Ralpha mRNA relative to other mRNAs (c-myc, beta-actin, IL2R beta, IL-6) for up to 12h after PHA stimulation, than did lymphocytes treated with a control oligomer of similar composition but different sequence. Nuclear run-on studies demonstrated that the rate of IL2R alpha mRNA synthesis relative to c-myc and beta-actin was also selectively diminished by treatment with 3'A-IL28p. These experiments suggest that transcription of individual genes can be selectively modulated in living cells by sequence specific collinear triplex formation in regulatory enhancer sequences.

Base Sequence↗
PubMed1991

Phased cis-acting promoter elements interact at short distances to direct avian skeletal alpha-actin gene transcription.

Recently, site-directed mutagenesis uncovered four positive cis-acting elements in the 5' promoter region of the chicken skeletal alpha-actin gene that directs myogenic tissue-restricted expression. In this study, interactions between the four promoter sites were examined by means of a series of insertion mutations that increased the linker region between adjacent elements by roughly half or complete DNA helical turns. Unexpectedly, transcriptional activity for all three sets of linker mutants, as assayed with a chloramphenicol acetyltransferase reporter gene, was found to vary in a fashion resembling a damped sinusoid with a period of roughly 10 base pairs, where the sinusoidal maxima appeared when length was increased by half-integral number of helix turns. We present a model which states that in the undistorted wild-type 5' flanking sequence, linker domains position each of the four promoter sites on the helix face opposite that of its immediate neighbors; when any of the three linkers is increased by approximately a half-integral number of helix turns, pairs of neighboring promoter sites are brought into alignment. We propose that this is the required orientation for inducing skeletal muscle-specific promoter activity, achieved in the wild-type promoter as a result of protein-induced torsional deformation.

Actins↗
PubMed1991

Effects of substrate texture and curvature on the morphology of cultured cells.

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.

Analog-Digital Conversion↗
PubMed1990

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending.

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.

Animals↗
PubMed1990

A photochemical method to map ethidium bromide binding sites on DNA: application to a bent DNA fragment.

It is shown that, when irradiated in the visible, ethidium bromide (EB) engages in direct photochemistry with its DNA binding site. At the photochemical end point, an average of one single-strand break is produced per bound EB molecule in a reaction which also bleaches the dye chromophore. Using high-resolution electrophoresis, we have mapped the distribution of EB photocleavage sites on DNA, at one-base resolution. It is argued that because the photocleavage is stoichiometric, the resulting pattern is similar to, if not identical with, the local distribution of EB binding affinity. When interpreted in the context of the extensive thermodynamic and structural data which are available for EB, a binding distribution of that kind can be used to infer details of DNA structure variation within the underlying helix. As a first application of the method, we have used EB to probe the structure of a 265 bp fragment of DNA, which had been described as being bent as the result of a periodic array of oligo(A) segments [Kitchin et al. (1986) J. Biol. Chem. 261, 11302]. The EB mapping data provide evidence that the oligo(A) elements in this fragment assume a local secondary structure which is different than that assumed by isolated ApA nearest neighbors and that the ends of the oligo(A) elements comprise a junctional domain with EB binding properties which differ from those of the oligo(A) element or of random-sequence DNA.

Adenine Nucleotides↗
PubMed1989

DNA flexibility variation may dominate DNase I cleavage.

In a previous experimental study, we proposed that the bending and torsional stiffness of DNA display a systematic sequence dependence. Subsequently, we developed an elastic strain model to quantify the sequence dependence of the bending and torsional rigidity in terms of nearest neighbor interactions and used that model to analyze the sequence dependence of the 434 repressor binding to its operator. The analysis presented here shows that, in the absence of significant local variation of DNA secondary structure, DNase I cleavage is strongly correlated with local variation in the bending flexibility as calculated from our elastic strain model and that the agreement is also quantitatively significant. It is proposed that analysis using elastic strain models will provide a preliminary set of biochemical and chemical tools to explore the relation between DNA flexibility and the binding of other proteins.

Base Sequence↗