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M H Caruthers

Publications and source records attributed to M H Caruthers.

At least 37 records · Page 2Linked to original sources
PubMed1990

Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding.

A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.

Amino Acid Sequence↗
PubMed1990

Antibodies specific for branched ribonucleic acids.

A chemically synthesized branched tetranucleotide, G3'p5'A [2'p5'G]3'p5'C corresponding to the consensus sequence at the branch point in introns undergoing RNA splicing, was used as a hapten to elicit antibranch antibodies. Binding assays with 32P-labeled hapten and unlabeled structurally related haptens indicated that the antibodies are highly specific for the branch structure and have some specificity for the A2'p5'G sequence at the branch point, but have essentially none for a variety of other 2'p5' or 3'p5' dinucleotides or for the linear trinucleotide G3'p5'A3'p5'C. Purification of these antibodies by binding to A2'p5'G covalently linked to Sepharose followed by covalent attachment of the purified antibodies to protein A-Sepharose has provided an adsorbent that immunospecifically retains branched oligonucleotides as well as branched introns released from RNAs during in vitro splicing.

Animals↗
PubMed1987

DNA functional groups required for formation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter. Identification via base analog substitutions.

Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed. Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition. Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role. Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.

Bacteriophage lambda↗
PubMed1986

Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability.

A solid-phase method using a phosphoramidite approach is described for synthesis of oligoribonucleotides. The method was used to synthesize pairs of oligomers with identical nearest neighbors but different sequences. Comparison of thermodynamic parameters for these pairs provides a test of the nearest-neighbor hypothesis for prediction of helix stability. In general, pairs of sequences with identical nearest neighbors have enthalpy and entropy changes for helix formation that differ by 8% on average, delta Go37 that differ by 6% on average, and melting temperatures within 0-5 degrees C of each other. These limits are typical of the accuracy that should be expected from nearest-neighbor predictions of RNA helix stability. UCAUGA and UGAUCA have the same nearest neighbors but melting temperatures that differ by 7 degrees C. This suggests some sequences will not be approximated well by the nearest-neighbor model.

Base Sequence↗
PubMed1986

Chemical synthesis of branched RNA.

A branched tetranucleotide consisting of adenosine linked 2' and 5' to guanosine and 3' to cytidine was synthesized from appropriately protected nucleoside phosphoramidites as synthons. The product was characterized enzymatically.

Chemical Phenomena↗
PubMed1986

Free energy contributions of G.U and other terminal mismatches to helix stability.

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.

Base Composition↗
PubMed1986

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids.

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.

Calorimetry↗
PubMed1986

Base substitution mutants of the lac operator: in vivo and in vitro affinities for lac repressor.

16 single-site mutations and a 1-bp deletion in the lac operator have been cloned and examined with regard to repressor binding. A 13-bp, central 'core' operator sequence, bp 5-17 of the natural operator, was also synthesized and cloned. Repressor affinity was assessed in vivo by quantitating the level of beta-galactosidase activity resulting from chromosomal operon derepression and in vitro by measuring the stability of repressor-operator complexes. Our results support the general conclusion that the repressor-operator interaction is asymmetric, particularly across the center of the operator sequence, with little or no specific contact at position 12. Some sequence changes in the right side of the operator markedly reduced repressor affinity, indicating that although binding to this half of the sequence has been suggested to be less important than the left half, it still significantly contributes to the binding affinity.

Cloning, Molecular↗
PubMed1986

Improved free-energy parameters for predictions of RNA duplex stability.

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in delta GO at 37 degrees C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5 degrees C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G X U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.

Hydrogen Bonding↗
PubMed1985

Gene synthesis machines: DNA chemistry and its uses.

Deoxyoligonucleotides can now be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid-phase synthesis on silica-based supports and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and DNA segments containing more than 100 deoxynucleotides.

Base Sequence↗
PubMed1985

Binding studies of SV40 T-antigen to SV40 binding site II.

SV40 T-Antigen binding site II was synthesized, cloned and analyzed for its ability to bind purified SV40 T-antigen. We report the binding constant of T-antigen for isolated site II. Using a filter binding assay the calculated binding constant was 6-8 fold less efficient than site I previously reported. Binding constants were calculated using two methods. The first was a direct calculation using a protein titration curve (KD). The second was by the ratio of measured association and dissociation rates. Both methods gave similar constants. Protection studies with SV40 T-antigen on the T-antigen binding sites in the wild-type array demonstrated that the binding constants of site I and site II are similar to those calculated for the individual sites. These results demonstrate that SV40 T-antigen does not bind cooperatively to sites one and two as earlier believed and are in agreement with recent observations emanating from several laboratories.

Antigens, Polyomavirus Transforming↗
PubMed1985

Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.

A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.

Bacteriophage lambda↗
PubMed1985

lac Up-promoter mutants with increased homology to the consensus promoter sequence.

Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.

Base Sequence↗
PubMed1984

Interaction of AD2+D2 protein and simian virus 40 large T antigen with the large tumor antigen binding site I.

In a lytic infection of a permissive host by SV40, the large tumor antigen (T antigen), which is a product of early transcription of the SV40 A gene, has been previously shown to autoregulate its own transcription by binding to SV40 DNA. The DNA region to which T antigen binds most tightly was synthesized and subsequently introduced into the bacterial plasmid pUC8. The interaction of SV40 T antigen with the DNA duplexes, derived from both chemical synthesis and the recombinant plasmid, were examined by using nitrocellulose filter binding assays. An SV40-adenovirus hybrid protein, AD2+D2 protein, was also tested. The SV40 T antigen was found to bind more tightly than the hybrid protein. Kinetic assays demonstrated that the association rates for the two proteins with the DNA binding site were equivalent; however, once formed, the T antigen-DNA complex dissociated more slowly than the AD2+D2 protein-DNA complex.

Antigens, Viral, Tumor↗
PubMed1984

Oligonucleotide mutagenesis of the lacPUV5 promoter.

Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter. Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site. The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.

Base Composition↗