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J Klysik

Publications and source records attributed to J Klysik.

28 records · Page 2Linked to original sources
PubMed1984

Intervening sequences in human fetal globin genes adopt left-handed Z helices.

The large intervening sequences ( IVS2 ) of three human fetal globin genes contain tracts of alternating purine-pyrimidine sequences approximately 40-60 base pairs in length which adopt left-handed Z DNA helices under the influence of negative supercoiling. The amount of negative supercoiling (approximately 0.045) required for the right- to left-handed transitions is within the physiological range. The structural aberrations between the right- and left-handed helices were mapped by sequencing the S1 nuclease cleavage sites. Two-dimensional gel electrophoretic analyses of the supercoil-induced relaxation served to characterize the type and length of left-handed structure. Furthermore, binding studies with several types of antibodies confirmed the presence of left-handed helices. Since these simple sequences appear to be hotspots for recombination and gene conversion, unusual DNA conformations may participate in genetic expression.

Base Sequence↗
PubMed1983

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.

Cytosine↗
PubMed1983

Conformational flexibility of junctions between contiguous B- and Z-DNAs in supercoiled plasmids.

The junctions between contiguous right- and left-handed helical DNA segments in supercoiled plasmids are shown to exhibit both sequence- and superhelical-dependent conformational flexibility. The nonalternating (purine-pyrimidine) tetranucleotide d(G-A-T-C), which is imbedded within a (dC-dG) tract and exists as a junction at low-density values, may be adopting a left-handed helix under the influence of higher negative supercoiling. The effect of supercoiling on the transition from a right- to a left-handed state for (dC-dG)n inserts, where n = 16, 13, and 5, also was evaluated. Only moderate amounts of superhelicity (-0.03 to -0.075) are required for formation and stabilization of left-handed DNA even for the (dC-dG) region of 10 dC X dG base pairs. Finally, we demonstrate that junction formation within a BamHI restriction site causes a drastic inhibition (greater than 80%) of cleavage by this restriction endonuclease.

DNA Restriction Enzymes↗
PubMed1983

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.

DNA↗
PubMed1982

Left-handed Z-DNA is induced by supercoiling in physiological ionic conditions.

In physiological ionic conditions (200 mM NaCl), the (dC-dG)16 and (dC-dG)13 blocks in plasmid pRW751 are in a left-handed state when the negative superhelical density of the plasmid is greater than 0.972. As the salt concentration decreases or when (dmC-dG) sequences are present, less negative supercoiling is required to induce the right- to left-handed DNA transition. Furthermore, the single strand-specific nuclease, S1, recognizes and cleaves aberrant structural features at the junction between neighbouring right- and left-handed DNA regions.

DNA, Superhelical↗
PubMed1982

Junction between Z and B conformations in a DNA restriction fragment: evaluation by Raman spectroscopy.

Raman vibrational spectra were obtained from two DNA restriction fragments and the DNA polymer (dG-dC)n . (dG-dC)n in 0.01 and 4.5 M NaCl. One fragment contained 95 base pairs (bp) of the Escherichia coli lactose operator-promoter region (95-bp fragment). The other fragment consisted of the 95-bp region flanked by 26 and 32 bp of dC-dG sequences and BamHI ends (157-bp fragment). In 0.01 M NaCl all three DNAs have Raman spectra characteristic of a right-handed B conformation. The high salt spectrum of the 95-bp fragment is also characteristic of a B conformation. However, the spectrum of the 157-bp fragment in 4.5 M NaCl shows major intensity changes from the 0.01 M NaCl spectrum. These changes are also observed in the high salt spectra of (dG-dC)n . (dG-dC)n and are correlated with the presence of a left-handed Z conformation. Comparisons between the high salt Raman spectra of the 157-bp fragment and spectra calculated from (dG-dC)n . (dG-dC)n and the 95-bp fragment indicated that essentially all of the dC-dG regions in the 157-bp fragment are in the Z conformation and a large part (approximately 80%) of the 95-bp region no longer has a B-type backbone vibration. However, this non-B-DNA-like character of the central region is not indicated by base vibrations.

Base Composition↗
PubMed1977

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).

Animals↗
PubMedDate not supplied

A 15-base acridine-conjugated oligodeoxynucleotide forms triplex DNA with its IL-2R alpha promoter target with greatly improved avidity.

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.

Acridines↗