Biomedical subjects

R H Durland

Publications and source records attributed to R H Durland.

11 recordsLinked to original sources

PubMed1995

Azole substituted oligonucleotides promote antiparallel triplex formation at non-homopurine duplex targets.

The ability of certain azole substituted oligodeoxy-ribonucleotides to promote antiparallel triple helix formation with duplex targets having CG or TA interruptions in the otherwise homopurine sequence was examined. 2'-Deoxyribonucleosides of the azoles, which include pyrazole, imidazole, 1,2,4-triazole and 1,2,3,4-tetrazole were synthesized using the stereo-specific sodium salt glycosylation procedure. These nucleosides were successfully incorporated using solid-support, phosphoramidite chemistry, into oligonucleotides designed to interact with the non-homopurine duplex targets. The interaction of these modified oligonucleotides with all four possible base pairs was evaluated and compared to similar data for a series of natural oligonucleotides. The oligonucleotides containing simple azoles enhanced the triplex forming ability considerably at non-homopurine targets. Binding of these modified oligonucleotides to duplex targets containing TA inversion sites was particularly noteworthy, and compare favorably to unmodified oligonucleotides for binding to duplex targets containing CG as well as TA base pairs. The selectivity exhibited by certain azoles is suggestive of base pair specific interactions. Thus, the azoles evaluated during this study show considerable promise for efforts to develop generalized triplex formation at non-homopurine duplex sequences.

Azoles↗

PubMed1995

Incorporation of 2'-deoxy-6-thioguanosine into G-rich oligodeoxyribonucleotides inhibits G-tetrad formation and facilitates triplex formation.

An efficient and expeditious method for the synthesis of S6-(cyanoethyl)-N2-isobutyryl (or trifluoroacetyl)-2'-deoxy-6-thioguanosine (7 and 2) from 2'-deoxyguanosine (G) has been developed. Compound 7 has been incorporated into several G-rich triple-helix-forming oligonucleotides (TFOs) using solid-support, phosphoramidite chemistry. The purified oligonucleotides containing 2'-deoxy-6-thioguanosine (S6-dG) residues in the place of G have been characterized by nucleoside composition analysis. These modified TFOs have been shown to be stable in aqueous, as well as buffered, solutions normally used to assay triple-helix formation. It has also been demonstrated that partial incorporation of S6-dG is effective in inhibiting the formation of G tetrads in G-rich oligodeoxyribonucleotides, thus facilitating triple-helix formation in potassium-containing buffers.

Base Sequence↗

PubMed1994

Binding of T and T analogs to CG base pairs in antiparallel triplexes.

The goal of this study was to address antiparallel triplex formation at duplex targets that do not conform to a strict oligopurine.oligopyrimidine motif. We focused on the ability of natural bases and base analogs incorporated into oligonucleotide third strands to bind to so-called CG inversions. These are sites where a cytosine base is present in an otherwise purine-rich strand of a duplex target. Using a 26-base-triplet test system, we found that of the standard bases, only thymine (T) shows substantial binding to CG inversions. This is quantitatively similar to the report of Beal and Dervan [Science (1991), 251, 1360-1363]. Binding to CG inversions was only slightly weaker than binding to AT base pairs. Binding of T to CG inversions was also evaluated in two other sequences, with qualitatively similar results. Six different analogs of thymine were also tested for binding to CG inversions and AT base pairs. Significant changes in affinity were observed. In particular, 5-fluoro-2'-deoxyuridine was found to increase affinity for CG inversions as well as for AT base pairs. Studies with oligonucleotides containing pyridin-2-one or pyridin-4-one suggest that thymine O4 plays a critical role in the T.CG interaction. Possible models to account for these observations are discussed.

Base Composition↗

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

Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria.

Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described. The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts. One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase. The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model. Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.

Bacterial Proteins↗

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↗

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↗

PubMed1990

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.

Bacterial Proteins↗

PubMed1990

Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers.

Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.

Alleles↗

PubMed1987

The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa.

The trfA gene of the broad-host-range plasmid RK2 encodes two proteins of 43- and 32-kDa by initiating translation at either of two in-phase AUG codons in a single open reading frame. At least one of these proteins is essential for replication of RK2 derivatives. In order to study the role of the 43-kDa protein, Bal31 deletions into the 5' end of the trfA gene were constructed and incorporated into minimal RK2 replicons. When examined in Escherichia coli, replication and maintenance properties of plasmids encoding only the 32-kDa protein were indistinguishable from those of plasmids encoding both the 43- and the 32-kDa proteins. In four other gram-negative hosts deletion of sequences encoding only the 43-kDa protein did not have a substantial effect on plasmid establishment or stable maintenance. However, in Pseudomonas aeruginosa, deletion of 43-kDa coding sequences greatly reduced the efficiency of plasmid maintenance, suggesting a host-specific role for the 43-kDa TrfA protein in RK2 replication.

Bacterial Proteins↗

PubMedDate not supplied

Selective binding of pyrido[2,3-d]pyrimidine 2'-deoxyribonucleoside to AT base pairs in antiparallel triple helices.

Triple helix-forming oligonucleotides (TFOs) offer the potential to specifically modulate expression of gene in a sequence dependent manner. TFOs containing G and T residues that bind to duplex DNA, forming a series of GGC and TAT base triplets, have been well studied. It has been observed that T is relatively nonspecific in that it binds with similar affinity to AT, GC, and CG base pairs. This may significantly reduce the specificity of a given TFO, leading to undesired effects on the expression of genes unrelated to the intended target. We have now prepared 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-pyrido[2,3-d]pyrimidine-2,7(8H )- dione (P) and incorporated it into TFOs using the solid-support, phosphoramidite chemistry. It has been demonstrated that a limited substitution of P for T in a G-rich 26-mer TFO can improve binding specificity for AT base pairs in antiparallel motif under certain conditions. The specificity exhibited by P is suggestive of base pair specific interactions that influence the binding strength and consequently enhance the potential therapeutic application of TFOs. However, the effect of substitution of P for T is dependent on the binding conditions, as well as the number of position of substitutions.

Adenine↗