Isolation of A-T-rich fragments from calf thymus DNA using the phenol method.

The action of phenol on the products of partial digestion of calf thymus DNA by K2 DNAase causes a loss of 3--5% of the material passing into the phenol phase. This part of DNA can be regained in the aqueous phase by lowering both the temperature and the ionic strength. Among oligonucleotides up to 7 monomers in length, those which are soluble in phenol do not contain guanine residues. Phenol-soluble DNA fragments of the molecular weight of an order of 2000--50,000 appeared to be composed mainly of adenosine phosphate. They also contain some thymine and only traces of guanine and cytosine. Some longer A-T-rich fragments, even up to 5-10(6) daltons, were repeatedly found in the phenol phase, but their base composition has not been determined yet. The method presented here was found very convenient for the isolation of relatively large A-T-rich DNA fragments. The property of DNA or its fragments to dissolve in phenol seems to be dependent on an adequate primary structure, probably similar to that of poly (dA-dT).

An intramolecular triplex structure from non-mirror repeated sequence containing both Py:Pu.Py and Pu:Pu.Py triads.

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.

Effects of 5 cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids.

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.

Escherichia coli single-stranded DNA-binding protein alters the structure of intramolecular triplexes in plasmids.

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.

Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+.

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).

Comparison of triple helix formation by polypurine versus polypyrimidine oligodeoxynucleotides when conjugated to a DNA intercalator.

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.

B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs.

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.

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide.

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.

Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids.

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.

The influence of recombinant human tumor necrosis factor-alpha, alone and in combination with cyclophosphamide or methotrexate, on leukemia L1210 and normal hematopoiesis in mice.

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)

Synthesis and expression in E. coli of the gene for human tumor necrosis factor (h alpha TNF).

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.

Inhibition of restriction endonuclease cleavage due to site-specific chemical modification of the B-Z junction in supercoiled DNA.

Structural distortions on the boundary between right-handed B and left-handed Z DNA segments in plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of chemical probes. Samples of supercoiled DNA were treated with the respective chemical probe, linearized with EcoRI and inhibition of BamHI (whose recognition sequence GGATCC lies on the boundary between the (dC-dG)n segments and the pBR322 nucleotide sequence) cleavage was tested. Treatment with osmium tetroxide in the presence of pyridine or 2,2'-bipyridine, respectively, resulted in a strong inhibition of the BamHI cleavage at both restriction sites, provided the (dC-dG)n segments were in the left-handed form. In the presence of 2,2'-bipyridine submillimolar concentrations of OsO4 (at 26 degrees C) were sufficient to induce the inhibition of BamHI. Chloroacetaldehyde was used as a probe reacting selectively with atoms involved in the Watson-Crick hydrogen bonding. Similarly as in the case of osmium tetroxide treatment of pRW751 with this agent resulted in the inhibition of BamHI cleavage. It was concluded that the B-Z junction regions in pRW751 contain few solitary bases with disturbed hydrogen bonding or non-Watson-Crick base pairs.

Concept of immunomics: a new frontier in the battle for gene function?

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.

Cruciform extrusion facilitates intramolecular triplex formation between distal oligopurine.oligopyrimidine tracts: long range effects.

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.

Both an altered DNA structure and cellular proteins are involved in protecting a triplex forming an oligopurine-rich sequence from Dam methylation in E. coli.

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.

Influence of DNA sequence on the formation of non-B right-handed helices in oligopurine.oligopyrimidine inserts in plasmids.

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.

Complex structural behavior of oligopurine-oligopyrimidine sequence cloned within the supercoiled plasmid.

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.

An integrated gene and SSLP BAC map framework of mouse chromosome 11.

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.

Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylated in vivo.

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.