Use of short oligonucleotides to screen cosmid libraries for clones containing G/C-rich sequences

We have developed a method to identify clones containing recognition sequences for enzymes that cut mammalian genomes infrequently by direct screening of genomic libraries. The degenerate oligonucleotide NNGCGGCCGCNN, in which the internal 8 bases correspond to the recognition sequence of Not I, was used to screen a cosmid library, and it led to a greater than 10-fold enrichment in the number of clones containing Not I sites. This technique permits the efficient identification of sufficient clones from a chromosome-specific library to allow the construction of a complete pulsed-field map of that chromosome and to assist in finding genes in genomic DNA.     

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.     

The binding of spermine to polynucleotides and complementary oligonucleotides at near physiological ionic strength

The binding of [14C] spermine to polynucleotides has been studied by equilibrium dialysis and the data analysed by Scatchard plots. The binding of spermine to poly(A) shows a binding site for 1 spermine/140 nucleotides when measured in 0.2M NaCl at 5 degrees C. Poly(C) also has a similar sites; on the other hand poly(U) and poly(G) each have a binding site for 1 spermine/12 nucleotides. The addition of complementary di- or trinucleotides to either poly(A) or poly(U) affects their ability to bind spermine, in particular the high affinity site on poly(A) is no longer detectable. The effect of spermine, spermidine and putrescine on the binding of polynucleotides to complementary di- and trinucleotides was also studied. Spermine markedly increased the binding of both ApA and of ApApA to poly(U) whereas spermidine and putrescine had very little effect. In contrast spermine had little effect on the binding of either UpU or UpUpU to poly(A). These results suggest that spermine binding to oligo- and polynucleotides is dependent on the particular nucleotide combination involved and that spermine may therefore be able to act selectively within cells.     

Crystallographic study of one turn of G/C-rich B-DNA

The DNA decamer d(CCAGGCCTGG) has been studied by X-ray crystallography. At a nominal resolution of 1.6 A, the structure was refined to R = 16.9% using stereochemical restraints. The oligodeoxyribonucleotide forms a straight B-DNA double helix with crystallographic dyad symmetry and ten base-pairs per turn. In the crystal lattice, DNA fragments stack end-to-end along the c-axis to form continuous double helices. The overall helical structure and, notably, the groove dimensions of the decamer are more similar to standard, fiber diffraction-determined B-DNA than A-tract DNA. A unique stacking geometry is observed at the CA/TG base-pair step, where an increased rotation about the helix axis and a sliding motion of the base-pairs along their long axes leads to a superposition of the base rings with neighboring carbonyl and amino functions. Three-center (bifurcated) hydrogen bonds are possible at the CC/GG base-pair steps of the decamer. In their common sequence elements, d(CCAGGCCTGG) and the related G.A mismatch decamer d(CCAAGATTGG) show very similar three-dimensional structures, except that d(CCAGGCCTGG) appears to have a less regularly hydrated minor groove. The paucity of minor groove hydration in the center of the decamer may be a general feature of G/C-rich DNA and explain its relative instability in the B-form of DNA.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.     

G-308A TNF-alpha polymorphism is associated with an increased risk of invasive cervical cancer

Cervical cancer is initiated by high-risk human papillomaviruses (HPV-16 and HPV-18), but an effective immune response may control the progression of this disease. Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine, that has been implicated in several cancers. In a case-control study, we evaluated the association between the G-308A TNF-alpha promoter polymorphism and the risk for invasive cervical cancer (ICC). TNF-alpha polymorphism was analyzed by PCR-RFLP and confirmed by sequencing. DNA was obtained from blood samples of 439 individuals, including 195 patients with ICC and 244 normal healthy controls. According to our results, women carrying the A allele present a twofold increased risk of developing ICC (p=0.006; OR=1.88; 95% CI [1.20-2.94]). In conclusion, our study suggests that the presence of the high producer allele -308A in the TNF-alpha gene appears to be associated with an increased risk for the development of ICC.     

Subcellular distribution of spermidine/spermine N1-acetyltransferase

The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was studied in L56Br-C1 cells treated with 10 microM N(1),N(11)-diethylnorspermine (DENSPM) for 24 h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM-treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM-induced apoptosis.     

Two mechanisms of spermidine/spermine N1-acetyltransferase-induction

The changes in activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme in polyamine degradation, were investigated to understand the mechanism of the induction of this enzyme in bovine lymphocytes. The activity of SAT was induced by stimulation with phytohemagglutinin (PHA), calcium ionophore A23187, sodium n-butyrate, or methylglyoxal bis(guanylhydrazone) (MGBG). When the cells were treated with a combination of PHA with either MGBG or butyrate, the increase in SAT was synergistic. However, the treatment of cells with both PHA and A23187 did not cause more induction of the enzyme activity than the stimulatory effects of each agent alone. The elevation in SAT caused by PHA or A23187 was inhibited by the simultaneous addition of 25 microM H-7, a protein kinase C inhibitor; the induction of the enzyme activity by MGBG or butyrate was slightly enhanced in the presence of H-7. In cells treated with a high concentration of O-tetradecanoylphorbol 13-acetate, which results in the breakdown of protein kinase C, PHA and A23187 did not give the maximum response, and MGBG slightly enhanced the enzyme activity. Dibutyryl cyclic AMP inhibited PHA-induced enzyme activity, but it stimulated MGBG- or butyrate-induced activity. Exposure to PHA or A23187 but not to MGBG or butyrate significantly increased the ornithine decarboxylase activity and DNA synthesis. These results showed that there were two different mechanisms of SAT induction. One is dependent on protein kinase C. The other one is independent of protein kinase C and is enhanced by cyclic AMP.     

Tissue-specific alternative splicing of spermidine/spermine N 1-acetyltransferase

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N ¹-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N ¹ ,N ¹¹ -diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.     

A charge-deficient analogue of spermine with chelating properties

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Interactions of mono spermine porphyrin derivative with DNAs

In this work, we have characterized the interactions of monospermine porphyrin derivative with calf thymus DNA (ct-DNA) and poly (dG-dC)₂ in both B and Z conformation. By several spectroscopic techniques (UV–vis, electronic circular dichroism and resonance light scattering), the binding modes of monospermine porphyrin derivative with different DNA sequences have been elucidated. In the presence of ct-DNA, the porphyrin binds along the external double helix as well as in the presence of B conformation of poly (dG-dC)₂. Whilst when the Z form of the poly (dG-dC)₂ is induced, a slight intercalation of the porphyrin between the basis has been detected.     

Interaction of oligonucleotides with cellular proteins

Oligonucleotides (ODNs) conjugated to 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODN-binding proteins in cells of different origin. The data obtained demonstrate that 68, 46, 38 and 28 kDa ODN-binding proteins are universal for tested cell lines.     

Inhibition of microRNA with antisense oligonucleotides

Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.     

The interaction of spermine and pentamines with DNA.

We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and 'melting' temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC). All pentamines caused aggregation of DNA at much lower concentrations than that of spermine. Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects. Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA. The biological role of pentamines in T. thermophilus appears to be related to their ability to promote DNA condensation at high temperature.