RESUMEN
At the beginning of the 21st century, biology will try to address the function of a large number of new genes. From the perspective of technologies applied today to functional genomics, this task appears to be more complex than the effort invested in the sequencing of the human genome. Conceptually, a high-throughput approach permitting correlation between newly discovered genes and functional properties of their protein products has yet to be developed. To address relationships between tens of thousands of genes and their cognate proteins, novel interdisciplinary technologies need to emerge. In this paper, a new idea of immunomics is presented and an experimental strategy is outlined to circumvent some of the restrictions associated with methodologies currently in use. It is proposed that cloned segments of genomic DNA are used for genetic immunization to obtain a large collection of antibodies, and to generate microarrays of these antibodies for tracing differentially expressed cellular proteins.
Asunto(s)
Anticuerpos/genética , Proteínas/genética , Proteínas/inmunología , Animales , Anticuerpos/inmunología , Genoma Humano , Humanos , Inmunización/métodos , Riesgo , Vacunas de ADN/genética , Vacunas de ADN/inmunologíaRESUMEN
Physical maps are important resources both in sequencing and in functional analyses of large genomes. Global contig-building approaches are regarded to be more efficient relative to the cumulative outcome of scattered and more localized physical mapping studies accompanying positional cloning. This work is part of an effort to assemble a complete physical map of mouse chromosome 11 in which selection of clones containing specific genetic markers from genomic libraries is the first step in the process. Using a previously developed strategy, we identified 361 bacterial artificial chromosomes (BACs) containing 88 gene markers. Since the linkage positions of markers chosen for these studies are known, the BAC framework obtained is anchored to the genetic map and represents about 13% of the length of the entire chromosome. Together with similar assignments of BACs generated previously using D11Mit markers (Cai et al., 1988, Genomics, 54: 387-397), 36-40% of the chromosome 11 is now assembled into contigs, and these contigs correlate through 51 clones carrying both gene and simple sequence length polymorphism markers.
Asunto(s)
Paseo de Cromosoma/métodos , Cromosomas Bacterianos/genética , ADN Recombinante/genética , Biblioteca de Genes , Marcadores Genéticos , Ratones/genética , Hibridación de Ácido Nucleico/métodos , Animales , Cromosomas/genética , ADN Bacteriano/genética , Ligamiento Genético , Sondas de OligonucleótidosRESUMEN
A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.
Asunto(s)
Acridinas/metabolismo , Adenina/metabolismo , Guanina/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/síntesis química , Purinas/metabolismo , Pirimidinas/metabolismo , Regulación de la Expresión Génica , Técnicas Genéticas , Sustancias Intercalantes/farmacología , Cinética , Modelos Moleculares , Pirroles/farmacologíaRESUMEN
Attachment of 6,9-diamino-2-methoxyacridine to the 5' end of a purine-rich oligodeoxynucleotide targeting a 15 bp oligopurine oligopyrimidine stretch in the promoter region of the interleukin-2 receptor alpha chain (IL-2R alpha) gene results in an approximately 500-fold increase in its triplex forming avidity as determined by both band shift assay and DMS footprinting (Kd lowered from 2.5 microM to 5 nM). This oligonucleotide participates in Mg(2+)-dependent three-stranded DNA formation in which it is oriented antiparallel relative to the purine strand of the target duplex as determined by acridine moiety sensitized photoreactivity with the target duplex DNA. The oligonucleotides used in these studies were synthesized with a 3-amino-2-hydroxypropyl group at the 3' end to protect against exonucleolytic degradation for future in vivo applications. The 3'-amino group underwent partial removal, probably during the NaOH deprotection step. Both the 3'-amino and the 3'-free forms of the oligo have the same binding avidity and specificity. The interaction of the third strand with its target is sequence specific and can be essentially abolished by a point G-->T transversion 4 bases away from the 3' end of the target oligopurine block or severely reduced by other mutations within the target duplex. Thus, the attachment of the acridine moiety to the 5' end of the oligonucleotide does not seem to substantially compromise the sequence specificity of binding. Additionally, the oligonucleotide composed of G and A nucleotides was found to be superior to the oligonucleotide containing G and T residues since the difference in avidity of binding to the same target site was 17-fold.
Asunto(s)
Acridinas/metabolismo , ADN/metabolismo , Sustancias Intercalantes/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Regiones Promotoras Genéticas , Receptores de Interleucina-2/genética , Acridinas/síntesis química , Acridinas/química , Sitios de Unión , ADN/química , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Potasio/farmacologíaRESUMEN
When the 4-bp Dam recognition sequence was placed between two d(GA)7 tracts, it became severely undermethylated in JM101 Escherichia coli cells compared to other Dam sequences in the same plasmid DNA. This site specific undermethylation was also detected on supercoiled molecules in vitro. Mutational analysis indicated that undermethylation is related to the capacity of the oligopurine tract to adopt the H-DNA conformation. In addition, chemical probing of the cells was consistent with a cellular protein bound to the DNA. Therefore it is likely that the combination of altered DNA conformation and a cellular protein leads to Dam-site protection. We also found that the site-specific undermethylation is detectable in certain E. coli strains only.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Purinas , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Secuencia de Bases , Sitios de Unión , Huella de ADN , Metilación de ADN , ADN Bacteriano/química , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Relación Estructura-ActividadRESUMEN
Biological applications of triplex forming oligonucleotides will require the development of oligomers with high avidity and specificity. We examined the binding enhancement resulting from intercalator conjugation to both parallel design (polythymidine T15) and antiparallel design (polypurine AG15, for binding a 15 base pair polypurine-polypyrimidine sequence in the IL-2R alpha gene enhancer) oligomers under various ionic strength and temperature conditions. Oligonucleotides were conjugated through a urea link to 6,9 diamino-3-methoxy acridine (to give T15C and AG15C). Intercalator conjugation dramatically enhanced the specific triplex binding avidity (Kd = 5 nM for AG15C and 275 nM for T15C at 25 degrees C, compared to 2 microM for AG15 and > 50 microM for T15 at 25 degrees C), without detectable binding to an inappropriate target sequence. Surprisingly, triplex formation with AG15C occurred at lower Mg2+ concentrations than with T15C. AG15 and AG15C showed rapid Mg2+ dependent self association, but not T15C or T15. T15C triplex formation occurred rapidly (completion in less than 4 min), while AG15C bound to its target sequence more slowly over 20-24 h. Thus, binding constants in the low nanomolar range are now achievable with intercalator conjugated polypurine antiparallel binding oligonucleotides, a prerequisite for biological applications of such agents.
Asunto(s)
Sustancias Intercalantes/química , Oligonucleótidos/química , Purinas/química , Pirimidinas/química , Secuencia de Bases , Tampones (Química) , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Interleucina-2/genética , Cinética , Magnesio/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , TemperaturaRESUMEN
A plasmid insert containing the (TA)7GATC(TA)7 inverted repeat and an adjacent (AG)7 tract adopts a cruciform structure at neutral pH. However, under acidic conditions the cruciform becomes readily disrupted in favor of a triplex. The (AG)7.(CT)7 duplex and one strand of the (TA)7GATC(TA)7 element are engaged in the three-stranded DNA formation, as determined using single-stranded DNA specific probes. The structure extrudes by displacing the (TA)7 strand adjacent to the (AG)7, and folding it back into the major groove of the (AG)7.(CT)7 duplex. This new variant of H-DNA is supercoil and pH dependent, requiring pH 6.1 or lower to form. The triplex is stabilized by T:A.T and A+:G.C triads. This unusual triad composition (50% of T:A.T and 50% of A+:G>C), that has not previously been observed in intramolecular triplexes, violates both the widely accepted division of triplexes into Py:Pu.Py or Pu:Pu.Py types and the requirement for mirror repeat symmetry.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , ADN Recombinante/química , Dietil Pirocarbonato , Electroforesis en Gel Bidimensional , Enlace de Hidrógeno , Datos de Secuencia Molecular , Plásmidos , Purinas/química , Pirimidinas/química , Sales (Química)RESUMEN
The ability of the Escherichia coli single-stranded DNA-binding protein (SSB) to recognize structural features associated with intramolecular triplex formation in oligopurine.oligopyrimidine (pur.pyr) inserts in recombinant plasmids was evaluated. The SSB protein binds to supercoiled plasmids and causes a site-preferential increase in OsO4 reactivity of the pyrimidine strand involved in the formation of the Hy-3 isomer of the triplex structure. The E. coli RecA protein showed no reaction with triplexes in similar studies. This behavior is consistent with SSB-mediated unpairing of the H-DNA-forming region.
Asunto(s)
ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Plásmidos , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ADN Superhelicoidal/química , ADN Superhelicoidal/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Tetróxido de Osmio/farmacología , Unión ProteicaRESUMEN
Two short d(AG)n tracts separated by either of two 40-base pair (bp) inverted repeats adopt a triple-stranded conformation (H-DNA) at the bottom of an extruded cruciform stem in negatively supercoiled plasmids at pH 4.5. Plasmids containing one d(AG)n adjacent to the inverted repeat or containing two d(AG)n tracts separated by a random sequence did not form the triplex structures. These conclusions were derived from chemical modification patterns and UV-sensitivity studies. Two-dimensional agarose gel electrophoresis revealed that the entire insert containing the cruciform and the triplex is locally unlinked. Moreover, a long range structural effect (over 40 bp of random sequence) of one (AG)7 block on the behavior of a second (AG)7 sequence was detected. This effect cannot be transmitted throughout a 50-bp segment of random sequence. A GenBank search revealed the frequent occurrence of short oligopurine blocks with an intervening random sequence of 17-40 bp.
Asunto(s)
ADN Superhelicoidal/química , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Plásmidos , Purinas , Secuencia de Bases , ADN Superhelicoidal/aislamiento & purificación , ADN Superhelicoidal/efectos de la radiación , Electroforesis en Gel de Agar , Concentración de Iones de Hidrógeno , Modelos Estructurales , Datos de Secuencia Molecular , Plásmidos/efectos de la radiación , Rayos UltravioletaRESUMEN
DNA oligonucleotides with appropriate sequences can form a stable duplex in which the two strands are paired in a parallel orientation instead of as the conventional antiparallel double helix of B-DNA. In parallel-stranded DNA (ps-DNA) base pairing is noncanonical with the glycosidic bonds in a trans orientation. The two grooves are equivalent. We have synthesized DNA duplexes consisting of a central parallel-stranded (dA)15.(dT)15 tract flanked by normal antiparallel regions, and ligated them into the pUC18 plasmid. The effect of negative supercoiling on the covalently closed circular molecules was studied by two-dimensional agarose gel electrophoresis and by chemical modification with OsO4-pyridine (Os,py) and diethylpyrocarbonate (DEPC). The following results were obtained: (i) The ps insert, and by inference ps-DNA in general, adopts a right handed helical form. (ii) Upon increasing the negative superhelix density (-sigma) to greater than 0.03 the 15 bp ps insert undergoes a major transition leading to a relaxation corresponding to a reduction in twist of approximately 2.5 helical turns. The transition free surgery is approximately kcal/mol. (iii) The chemical modification pattern of the resulting structure suggests that the purine strand folds back and associates with the pyrimidine strand, forming a novel intramolecular triplex structure consisting of d(A.A.T) base triplets. A model for the triplex conformation is proposed and its thermodynamic properties are analyzed by statistical mechanics.
Asunto(s)
ADN Circular/química , ADN Superhelicoidal/química , Poli dA-dT/química , Composición de Base , Secuencia de Bases , Dietil Pirocarbonato/química , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Tetróxido de Osmio/química , Plásmidos/genética , EspectrofotometríaRESUMEN
We investigated the influence of rh-TNF administered as a single agent or in combination with CY or MTX on the survival time of mice inoculated with lymphoid leukemia L1210 and the effects of similar treatment on normal hematopoiesis in mice. The MST of rh-TNF--treated mice was longer than that of control animals. The longest survivals were observed in mice treated with 250 and 275 micrograms/kg of rh-TNF. Groups of mice receiving a combination of rh-TNF at doses of 225 or 250 micrograms/kg and MTX lived longer than animals treated with these agents separately. We observed the longest survival time of mice treated with combined administration of rh-TNF at a dose of 250 micrograms/kg and CY, but survival time was not significantly prolonged compared with mice receiving only CY. Additional studies were performed to examine the influence of rh-TNF administered as a single agent or in combination with toxic doses of CY or MTX on the number of granulocytes, lymphocytes, erythrocytes with hematocrit values and hemoglobin concentration, and platelets in peripheral blood, and the number of mononuclear cells as well as multipotential stem cells (CFU-GEMM) in bone marrow. Rh-TNF caused dose-dependent suppression of mononuclear cells and multipotential stem cells in bone marrow. The addition of MTX to rh-TNF caused no enhanced suppression of any of the above mentioned hematological parameters. In contrast, the addition of CY to rh-TNF suppressed erythrocytes and hematocrit values, as compared with rh-TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ciclofosfamida/administración & dosificación , Leucemia L1210/tratamiento farmacológico , Metotrexato/administración & dosificación , Factor de Necrosis Tumoral alfa/administración & dosificación , Animales , Recuento de Células Sanguíneas , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Femenino , Hematopoyesis/efectos de los fármacos , Leucemia L1210/sangre , Leucemia L1210/patología , Ratones , Ratones Endogámicos DBARESUMEN
A gene coding for human tumor necrosis factor (h alpha TNF) has been assembled by ligating short oligodeoxyribonucleotides and cloning into plasmid vectors. These oligonucleotides were prepared by the modified phosphoramidite methodology using isopropoxyacetyl (IPA) as a protecting group for exoamino- functions of nucleosides. Gene was expressed in E. coli and the protein product was purified to homogeneity by ion-exchange chromatography.
Asunto(s)
Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Escherichia coli/genética , Expresión Génica , Variación Genética , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Factor de Necrosis Tumoral alfa/aislamiento & purificaciónRESUMEN
Four 25-nt long oligonucleotides containing dA and dT (D1, D2, D3, and D4) which are capable of forming parallel-stranded (ps) or antiparallel-stranded (aps) duplexes have been synthesized [Rippe, K., Ramsing, N. B., & Jovin, T. M. (1989) Biochemistry 28, 9536-9541]. In the present study, the OsO4-pyridine complex (Os,py), diethyl pyrocarbonate (DEPC), KMnO4, and the 1,10-phenanthroline-cuprous complex [(OP)2Cu+] were used to investigate the conformation-dependent reactivity of ps, aps, and single-stranded (ss) oligonucleotides. The products were analyzed by polyacrylamide gel electrophoresis with single-nucleotide resolution. The results confirm the duplex nature of the ps combinations of oligonucleotides and reveal structural differences in comparison with the aps molecules. Under conditions in which ss-DNA is substantially sensitive to Os,py, both the ps and aps duplexes are very unreactive. A similar result was observed with KMnO4 and DEPC, although with the latter reagent the modification pattern of the labeled strands D1* and D4* was slightly different for the parallel than for the antiparallel duplex. The (OP)2Cu+ complex efficiently cleaves the aps but not the ps duplex and shows a preference for TAT steps. We also tested the effect of monovalent and divalent cation concentrations on the chemical reactivity of the ps, aps, and ss species. Elevated NaCl concentration leads to a dramatic increase in the Os,py and KMnO4 modification of ss molecules and the ps, but not the aps, duplex. We attribute the apparent reaction with ps-DNA to a destabilization of this conformation under the conditions of reaction. In contrast, all reactions with DEPC are somewhat depressed at high salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Daño del ADN , Oligodesoxirribonucleótidos/química , Secuencia de Bases , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de los fármacos , Dietil Pirocarbonato/farmacología , Conformación Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Tetróxido de Osmio/farmacología , Fenantrolinas/farmacología , Poli dA-dT/química , Permanganato de Potasio/farmacologíaRESUMEN
Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites. Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain. Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation. Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo. Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation. E.coli host factors are involved in the observed phenomenon. This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector. Full methylation can be readily achieved in vitro. Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures.
Asunto(s)
ADN Bacteriano , Metiltransferasas/metabolismo , Conformación de Ácido Nucleico , Plásmidos , Polidesoxirribonucleótidos/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica) , Secuencia de Bases , Cloranfenicol/farmacología , Clonación Molecular , Enzimas de Restricción del ADN , Escherichia coli/genética , Proteínas de Escherichia coli , Cinética , Metilación , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Nucleótidos de Purina , Nucleótidos de PirimidinaRESUMEN
Human peripheral blood monocytes pretreated with human recombinant tumour necrosis factor alpha (rTNF) showed an enhanced ability to present a soluble antigen, a purified protein derivate of tuberculin, to autologous T lymphocytes as assessed by their increased proliferation in vitro. This enhancing activity was due to TNF and not impurities in TNF preparations as anti-TNF antibodies abolished this phenomenon. The rTNF-treated monocytes showed an increased expression of HLA-DR molecules and enhanced co-stimulatory activity in the murine thymocyte assay. Pretreatment of monocytes before an antigen pulse with anti-TNF mAb inhibited antigen presentation, which indicated that endogenously produced TNF was involved. These studies thus suggest that TNF acts in an autocrine fashion and enhances the ability of monocytes to present protein antigen. It is unclear at present whether this effect is due to the modification in antigen processing, expression of MHC class II molecules, or other factors (IL-1, IL-6, adhesion molecules, etc.) that are important for the induction of T cell response to a nominal antigen. The enhancement of the antigen presenting capacity of monocytes/macrophages may be the additional mechanism of pro-inflammatory activity of TNF.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Anticuerpos Monoclonales , Antígenos , Antígenos HLA-DR , Humanos , Técnicas In Vitro , Tuberculina/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).
Asunto(s)
Elementos Transponibles de ADN/efectos de los fármacos , ADN Superhelicoidal/efectos de los fármacos , Níquel/farmacología , Poli dA-dT/análisis , Polidesoxirribonucleótidos/análisis , ADN Superhelicoidal/análisis , Dietil Pirocarbonato , Endodesoxirribonucleasas , Conformación de Ácido Nucleico , Mapeo Nucleótido , PlásmidosRESUMEN
Synthetic sequence GATCC(AG)7ATCG(AT)4CG(AG)7 was cloned into plasmid and its structural behavior under the influence of supercoiling was analysed by chemical modification at variety of experimental conditions. It was found that this sequence adopts at least two different non-B conformations depending on -delta and pH values. Moreover, 12 nucleotide long non-pur.pyr spacer region separating two identical (AG)7 blocks does not provide a significant energy barrier protecting against unusual structures formation.
Asunto(s)
ADN Superhelicoidal , Conformación de Ácido Nucleico , Plásmidos , Nucleósidos de Purina , Nucleósidos de Pirimidina , Secuencia de Bases , Clonación Molecular , Dietil Pirocarbonato , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Mapeo Nucleótido , Tetróxido de Osmio , Relación Estructura-ActividadRESUMEN
Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.
Asunto(s)
ADN Superhelicoidal , Conformación de Ácido Nucleico , 5-Metilcitosina , Citosina/análogos & derivados , ADN , Distamicinas/farmacología , Enlace de Hidrógeno , Metilación , Conformación de Ácido Nucleico/efectos de los fármacos , Plásmidos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
A systematic study was conducted on seven recombinant plasmids harboring synthetic inserts which had all purines on one strand and all pyrimidines on the complementary strand (Pur.Pyr). The inserts ranged in G+C content from 100% [G19.C19] to 0% [A20.T20] with intermediate contents at 66% [(TCC)8.(GGA)8], 50% [(CT)12.(AG)12 and (TTCC)6.(GGAA)6], 33% [(TTC)8.(GAA)8], and 25% [(GAAA)6.(TTTC)6]. The specific reactions at the base pair level of these inserts with enzymatic (S1 and P1 nucleases) and chemical (bromoacetaldehyde, OsO4, diethyl pyrocarbonate, and dimethyl sulfate) probes were evaluated as influenced by pH, negative supercoiling, and ionic strength (NaCl). Supercoil-induced relaxation studies using two-dimensional gels also provided important conformational information. We conclude that the five inserts with 66-25% G+C adopt a non-B right-handed conformation which is stabilized by negative supercoiling. Low pH (pH values 4.5-5.0) tends to stabilize this structure but is not essential for its formation. Surprisingly, an end bias of reactivity from the center toward the 5'-end of the purine strand of these inserts was generally found for the enzymatic and chemical probes which was irrespective of the orientation of the insert in the pRW790 vector. An intramolecular triple-stranded model for the unusual structure of the insert accounts most favorably for these observations. Unexpectedly, the A20.T20 insert seems to remain in an orthodox right-handed B-conformation under all conditions tested. The G19.C19 insert does adopt a non-B right-handed structure as for the five inserts with 66-25% G+C, but the pattern of reactivities and hence its conformation is different.
Asunto(s)
Elementos Transponibles de ADN , ADN Bacteriano , Conformación de Ácido Nucleico , Plásmidos , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Superhelicoidal , Escherichia coli/genética , Datos de Secuencia Molecular , OligodesoxirribonucleótidosRESUMEN
Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.