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Biomedical subjects

D S Sigman

Publications and source records attributed to D S Sigman.

At least 37 records · Page 2Linked to original sources
PubMed1994

A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity.

Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif. Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors. Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot. One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor. A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct. This nuclease has an altered specificity of DNA binding in vitro. When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency.

Bacterial Proteins↗
PubMed1994

Oligonucleotide-directed nucleic acid scission by micrococcal nuclease.

"Sequence-dictated" scission of DNA can be achieved by tethering a fusion protein composed of glutathione S-transferase and attenuated micrococcal nuclease (MN) to a targeting oligonucleotide using Cibacron blue (CB) F3G-A. Deoxyoligonucleotides derivatized with this dye bind to the fusion protein in gel mobility shift assays. This binding scheme was successfully used to achieve site-specific scission of a single-stranded DNA substrate after hybridization with a CB-derivatized complementary oligonucleotide. Although covalently cross-linked hybrids of MN and oligonucleotides have been successfully used in the past to target nucleolytic activity, this novel scheme opens new possibilities for targeting and probing both DNA and RNA sequences by allowing the addition of the nuclease subsequent to hybridization.

Base Sequence↗
PubMed1994

Footprinting RNA-protein complexes following gel retardation assays: application to the R-17-procoat-RNA and tat--TAR interactions.

RNA-protein complexes isolated following a gel retardation assay can be footprinted within the gel matrix using the chemical nuclease activities of 4,7-dimethyl-, 5,6-dimethyl-, and 3,4,7,8-tetramethyl-1,10-phenanthroline-copper. These complexes are more reactive than 1,10-phenanthroline-copper but share its reaction preference for bulges and loops. The interaction of the coat protein of R-17 with its viral RNA target and tat- and tat-derived peptides with HIV TAR RNA have been studied. In both cases, the RNA sequence opposite a 2-3 nucleotide bulge are protected. Tat-derived peptides inhibit cleavage at sites which intact tat does not protect. These results are consistent with transcription studies which have suggested that truncation of tat increases nonspecific binding.

Amino Acid Sequence↗
PubMed1994

Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex. Potential for gene-specific inactivation reagents.

An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase. Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide. When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed. The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies.

Base Sequence↗
PubMed1994

Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex.

The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics. Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex [(2,9-Me2OP)2Cu+] and rifampicin, bind stably to the open-complex. (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres. Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP. Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex. Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter. The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.

Base Sequence↗
PubMed1994

DNA-binding proteins as site-specific nucleases.

DNA-binding proteins can be converted into site-specific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor groove-proximal amino acid to a cysteine residue using site-directed mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10-phenanthroline-copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high-resolution nuclear magnetic resonance and X-ray crystal structures, and to locate binding sites of proteins within genomes.

Catalysis↗
PubMed1993

A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes.

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.

Cross-Linking Reagents↗
PubMed1993

Sequence-specific scission of DNA by RNAs linked to a chemical nuclease.

RNAs linked to the chemical nuclease 1,10-phenanthroline-copper cut double-stranded DNA of complementary sequence. This cleavage reaction is applicable to all sequences and can be used to measure the distance between marker genes in base pairs, map the size of a transcription unit and define positions of chromosomal breakpoints.

Copper↗
PubMed1993

Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.

RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stranded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoallyl)-UTP as the sole source of UTP and can be readily modified with 1,10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-stranded DNAs are efficiently cleaved at multiple sites because 1,10-phenanthroline is incorporated at several uridines in the sequence. Sequence-specific double-stranded scission of duplex DNA can also be accomplished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displacement of one strand of DNA by the RNA of identical sequence. R loop-directed scission is the first method for DNA scission applicable to any sequence. A unique application of R loop-targeted nucleolytic scission, which relies on its ability to cut DNA at any sequence, is the determination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared from the two distinct sequences and used to cut the DNA fragment after R-loop formation. The size of the fragment liberated by these methods is a direct measure in base pairs of the distance between the two DNA sequences. For example, the distance separating two chicken delta crystallin (delta 1 and delta 2) genes has been confirmed as 24 kilobases by this method.

Animals↗
PubMed1993

Transforming the Escherichia coli Trp repressor into a site-specific nuclease.

The Escherichia coli Trp repressor has been converted into an operator-specific nuclease by alkylating cysteine-49, inserted by site-directed mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline. In the presence of copper ion and thiol, high yields (> 50%) of double-stranded breaks of DNA are observed after a 20-min reaction. The high cleavage efficiency of this derivatized protein (Trp repressor E49C-OP) can be attributed to the proximity of cysteine-49 to the minor groove, the site of the C-1H of the deoxyribose and the target of the oxidative nuclease activity of (1,10-phenanthroline)copper. Since sequence position 49 is close to the protein's C2 dyad axis and adjacent to the minor groove, Trp repressor E49C-OP reacts with the operator DNA near the binding site of this symmetry locus of the protein. The patterns of scission of the trpR, aroH, and trpEDCBA operators (a) confirm the orientation of the repressor to the operator predicted from the X-ray study of a cocrystal (Otwinowski et al., 1988) and (b) support the model for tandem binding of the repressor to the trpR, aroH, and trpEDCBA operators based on DNase I footprinting and methylation interference (Kumamoto et al., 1987). There are one, two, and three binding sites for the repressor on the trpR, aroH, and trpEDCBA operators, respectively. In addition to providing a novel approach to studying the interactions of DNA binding proteins, 1,10-phenanthroline-derivatized proteins such as Trp repressor E49C-OP may be useful as rare cutters in the analysis of high molecular weight DNAs, especially if their binding specificities can be altered.

Amino Acid Sequence↗
PubMed1992

1,10-Phenanthroline-copper, a footprinting reagent for single-stranded regions of RNAs.

The 1,10-phenanthroline-cuprous complex (OP-Cu) with hydrogen peroxide as a coreactant nicks the single-stranded loops and bulges of RNA stem-loop structures more rapidly than the double-stranded stems. This chemical nuclease is therefore a useful footprinting reagent for these regions and can be used to monitor both intramolecular and intermolecular hybridization of single-stranded domains. The formation of A-form structures characteristic of either RNA-RNA or RNA-DNA duplexes inhibits scission because it blocks the binding site of the coordination complex in single-stranded loops and not because the oxidatively sensitive hydrogens of the ribose moiety are blocked. The C-4' and C-1' hydrogens are accessible to solvent in A-structures.

Base Sequence↗
PubMed1991

Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeat.

Human immunodeficiency virus (HIV) gene expression is regulated by both general transcription factors and factors induced by activation of T lymphocytes such as NF-kappa B and the nuclear factor of activated T cells (NFAT). Within the HIV long terminal repeat (LTR), two purine-rich domains between nucleotides -283 and -195 have homology to a regulatory region found in the interleukin 2 promoter, which binds NFAT and other cellular factors. In the HIV LTR, this region has been demonstrated to have both positive and negative regulatory effects on HIV gene expression. In an attempt to clone genes encoding cellular factors that bind to these NFAT-like elements in the HIV LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to these binding motifs. A ubiquitously expressed cDNA encoding a 60-kDa protein, which we termed interleukin binding factor (ILF), binds specifically to these purine-rich motifs in the HIV LTR. This factor also binds to similar purine-rich motifs in the interleukin 2 promoter, through with lower affinity than to HIV LTR sequences. Sequence analysis reveals that the DNA binding domain of ILF has strong homology to the recently described fork head DNA binding domain found in the Drosophila homeotic protein fork head and a family of hepatocyte nuclear factors, HNF-3. Other domains found in ILF include a nucleotide binding site, an N-glycosylation motif, a signal for ubiquitin-mediated degradation, and a potential nuclear localization signal. These results describe a DNA binding protein that may be involved in both positive and negative regulation of important viral and cellular promoter elements.

Amino Acid Sequence↗
PubMed1991

New inhibitors of aldose reductase: anti-oximes of aromatic aldehydes.

Aldose reductase is an NADPH-dependent enzyme which catalyzes the reduction of glucose to sorbitol. Specific potent inhibitors of aldose reductase are of potential pharmacological use because elevated levels of sorbitol produced by this enzyme in lens, peripheral nerve, retina, and renal glomeruli may be responsible for the pathogenesis associated with chronic diabetes. These inhibitors could also serve as probes of the mechanism of action of aldose reductase. anti-Oximes of aromatic aldehydes (e.g., benzaldoxime and 4-fluorobenzaldoxime) have proved to be effective inhibitors of aldose reductase rivaling pharmacological agents currently used to inhibit this enzyme in vivo. The kinetic patterns of inhibition in which benzyl alcohol is used as the oxidizable substrate suggest that the inhibition is due to the formation of a stable ternary complex composed of aldose reductase, NADP+, and the anti-oxime. Analogus ternary complexes are formed at the active site of horse liver alcohol dehydrogenase which is also inhibited by anti-oximes of efficient substrates.

Alcohol Dehydrogenase↗
PubMed1991

Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease.

A 6 M guanidine-HCl/0.2 M EDTA solution was used to lyse and store whole blood specimens. DNA stored in guanidine-EDTA-blood (GEB) lysate was found to be undegraded after incubation at 37 degrees C for 1 month, suggesting that this represents an appropriate reagent for transport of blood samples from the field to a laboratory for analysis. Trypanosoma cruzi kinetoplast DNA in GEB lysate can be cleaved using the chemical nuclease, 1,10-phenanthroline-copper ion (OP-Cu2+). This procedure liberates linearized minicircle molecules from network catenation, distributing them throughout the lysate, and allowing a small aliquot of the original lysate to be analyzed by PCR amplification. This increases the sensitivity of the method dramatically for the detection of small numbers of trypanosomes in a large volume of blood. DNAs isolated from aliquots of T. cruzi-positive GEB lysates were polymerase chain reaction (PCR)-amplified with 3 sets of T. cruzi-specific kDNA minicircle primers, yielding the 83-bp and 122-bp conserved region fragments and the 330-bp variable region fragments. The PCR products were analyzed by gel electrophoresis and/or hybridization. Results indicate that a single T. cruzi cell in 20 ml of blood can be detected by this method. Blood samples from several chronic chagasic patients were tested. Amplification of T. cruzi kDNA minicircle sequences was obtained in al cases, even when xenodiagnosis was negative. This PCR-based test should prove useful as a replacement or complement for xenodiagnosis or serology in clinical and epidemiological studies of chronic Chagas' disease.

Animals↗
PubMed1990

Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymerase.

The nuclease activity of the copper complex of 5-phenyl-1,10-phenanthroline (5-phi-OP-Cu) detects conformational changes in the lac UV-5 promoter caused by E. Coli RNA polymerase. The template strand in melted regions of initiation complexes upstream of the site of nucleotide triphosphate incorporation is very reactive. In open and elongation complexes, downstream scission sites (e.g. +4 and +5 for the open complex) on both strands are observed. The patterns of both downstream cutting sites suggest an atypical double helix at the leading edge of the transcription bubble.

DNA↗
PubMed1990

Sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.

A statistical analysis of a data set composed of over 1600 scission events of DNA produced by the 2:1 1,10-phenanthroline-copper complex (OP-Cu) has demonstrated that the nucleotide 5' to the site of phosphodiester bond scission is a primary influence in the kinetics of cleavage at any sequence position. The scission was less affected by the 3' neighbor. For each of the sixteen possible dinucleotides, a kinetic parameter can be computed reflecting scission at the 3' nucleotide. When used to predict the scission pattern of a DNA sequence not part of the present data set, correlation coefficients of about 0.6 between predicted and observed patterns were obtained.

Base Sequence↗