Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.

Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS-3A-gene. This factor, designated GT-1, binds to two short sequences (boxes II and III) in the -150 region that are known to function as light-responsive elements (LREs) in transgenic tobacco. Binding of GT-1 to homologous sequences further upstream (boxes II and III in the -220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted. In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT-binding. Single Gs present in boxes III and III are also important. Since GT-1 is present in nuclear extracts from leaves of light-grown and dark-adapted pea plants, its regulatory role does not depend on de novo synthesis. Thus if GT-1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light.     

Binding sites for two novel phosphoproteins, 3AF5 and 3AF3, are required for rbcS-3A expression.

Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding sites for the nuclear factor GT-1 within the -166 deleted promoter of the ribulose-1,5-bisphosphate carboxylase-3A (rbcS-3A) gene, suggested that GT-1 might act in concert with an additional protein to confer light-responsive rbcS-3A expression. In this work, S1 analysis of RNA isolated from transgenic tobacco plants carrying mutant rbcS-3A constructs led to the identification of two short sequences located at the 5' and 3' ends of box III that are required for expression. These two sequences serve as binding sites for two novel proteins, 3AF5 and 3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and 3AF3 showed that complexes with faster mobilities were formed using nuclear extracts prepared from dark-adapted plants compared with those from light-grown tobacco plants. Phosphatase treatment of extracts from light-grown plants resulted in the formation of complexes with faster mobility. Although the binding of 3AF3 to its target site is dependent upon phosphorylation, the binding of 3AF5 does not appear to be affected by its phosphorylation state. These results suggest that the phosphorylated forms of both 3AF5 and 3AF3 are required for -166 rbcS-3A expression but that the mechanisms differ by which phosphorylation regulates the activities of 3AF5 and 3AF3.     

Leishmania spp.: cellular levels and synthesis of polyamines during growth cycles

Polyamines were extracted from five different leishmanial strains (Leishmania sp., L. tropica major, L. mexicana, and two L. donovani isolates) and identified as pulrescine, spermidine, and spermine by thin-layer chromatography and mass spectrometry. These sensitive methods were also used to demonstrate the conversion of radioactive putrescine into spermidine and spermine. As in other types of cells, polyamine levels fluctuated during the growth cycle, maximal levels being attained during the logarithmic growth phase. In the five leishmanial strains, which were members of four different serotypes, the spermidine—putrescine ratios also varied, and in two strains of the same serotype, polyamine ratios were practically identical, suggesting that polyamine characteristics might serve as a further criterion for strain identification and classification.     

Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA

The effect of a series of aminooxy analogues of the biogenic polyamines spermidine and spermine on the conformation of calf thymus DNA is studied. These new molecules are isosteric and charge insufficient analogues that are suitable to study the roles of both charge distribution and structural requirements in the molecular physiology of the biogenic polyamines. They are also evidenced as useful tools to inhibit polyamine biosynthesis and cell growth. Circular dichroism (CD) spectra of solutions containing DNA and the aminooxy analogues at different concentrations (100-1000 microM) and different pH values, (5-7.5) are recorded. We use both sonicated and highly polymerized calf thymus DNA. The CD spectra of sonicated DNA showed the formation of Psi-DNA, a highly ordered aggregated structure similar to liquid crystals, in the presence of the aminooxy analogues. Aggregation induced by an aminooxy derivative of spermine is followed by DNA collapse when increasing the polyamine concentration. The features of Psi-DNA are not detected for highly polymerized DNA. Temperature melting measurements support a high degree of structural order of the aggregates. The CD experiments indicate that dications are unable to induce major changes on the macromolecular structure of DNA. In addition, aggregation is only observed when the trimethylene moiety is present between two adjacent positive charges. The observed differences among the CD spectra of DNA solutions with different aminooxy derivatives of spermidine indicate different roles for different amino groups of this biogenic polyamine when interacting with DNA. Our results support the idea that aminooxy analogues can be used as good models in studying the physiological functions of biogenic polyamines.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

Level of expression of the tomato rbcS-3A gene is modulated by a far upstream promoter element in a developmentally regulated manner.

By Agrobacterium-mediated transformation we have demonstrated that a 1.10-kilobase promoter sequence from the tomato rbcS-3A gene confers light-inducible and organ-specific expression upon fusion to the bacterial chloramphenicol acetyltransferase gene. A biphasic expression profile was obtained by 5' deletion analysis of this promoter, indicating the presence of both positive and negative regulatory elements. A severe reduction in the level of expression was observed when the 5'-terminal 90 base pairs were deleted from the 1.10-kilobase promoter. DNA sequence elements responsible for light inducibility and organ specificity of the gene reside within the -374 base pairs of the proximal part of the promoter and the sequences spanning from -374 to -205 are essential for promoter function. The DNA sequences upstream from -374 modulate the level of expression in leaf tissue; this modulation is under developmental control.     

Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA

DNA condensation, precipitation, and aggregation are related phenomena involving DNA-DNA interactions in the presence of multivalent cations, and studied for their potential implications in DNA packaging in the cell. Recent studies have shown that the condensation/aggregation is a prerequisite for the cellular uptake of DNA for gene therapy applications. To elucidate the ionic and structural factors involved in DNA aggregation, we studied the precipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) triplex DNA in the presence of the tetravalent polyamine spermine using a centrifugation assay, Tm measurements, and CD spectroscopy. The ability of spermine to provoke DNA precipitation was in the following order: triplex DNA > duplex DNA > single-stranded DNA. In contrast, their resolubilization at high polyamine concentrations followed a reverse order. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. Tm data indicated the DNA stabilizing effect of spermine even in the resolubilized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and triplex DNA, with the duplex form regaining the B-DNA conformation at high concentrations (approximately 200 mM) of spermine. The triplex DNA, however, remained in a Psi-DNA conformation in the resolubilized state. Chemical structural specificity effects were exerted by spermidine and spermine analogues in precipitating and resolubilizing sonicated calf thymus DNA, with N4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine having the maximal difference in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. The effect of the cationicity of polyamines was evident with the pentamines being much more efficacious than the tetramines and triamines. These results provide new insights into the mechanism of DNA precipitation by polyamines, and suggest the importance of polyamine structure in developing gene delivery vehicles for therapeutic applications.     

Study by optical spectroscopy and molecular dynamics of the interaction of acridine-spermine conjugate with DNA

We report a spectroscopic and theoretical study of the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound. Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumour drugs.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

The growth promoter spermine interacts specifically with dermatan sulfate regions that are rich inl-iduronic acid and possess antiproliferative activity

We have investigated interactions between spermine, a member of the growth promoting polyamine family, and various glycosaminoglycans. By using gel chromatography and equilibrium dialysis experiments we found that spermine binds tol-iduronic acid-rich dermatan sulfate (K _d, approximately 3.9×10^–4 M) with an affinity similar to that between spermine and DNA. By digesting spermine-dermatan sulfate complexes with chondroitin ABC lyase, the formation of oligosaccharide fragments (tetra-to-decasaccharides) was demonstrated by polyacrylamide gel electrophoresis. Chondroitin sulfate, which is deficient inl-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of thel-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.     

Spermine and gene methylation: a mechanism of lifespan extension induced by polyamine-rich diet

The polyamines spermidine and spermine are synthesized in almost all organisms and are also contained in food. Polyamine synthesis decreases with aging, but no significant decrease in polyamine concentrations were found in organs, tissues, and blood of adult animals and humans. We found that healthy dietary patterns were associated with a preference for polyamine-rich foods, and first reported that increased polyamine intake extended the lifespan of mice and decreased the incidence of colon cancer induced by repeated administration of moderate amounts of a carcinogen. Recent investigations have revealed that changes in DNA methylation status play an important role in lifespan and aging-associated pathologies. The methylation of DNA is regulated by DNA methyltransferases in the presence of S-adenosylmethionine. Decarboxylated S-adenosylmethionine, converted from S-adenosylmethionine by S-adenosylmethionine decarboxylase, provides an aminopropyl group to synthesize spermine and spermidine and acts to inhibit DNMT activity. Long-term increased polyamine intake were shown to elevate blood spermine levels in mice and humans. In vitro studies demonstrated that spermine reversed changes induced by the inhibition of ornithine decarboxylase (e.g., increased decarboxylated S-adenosylmethionine, decreased DNA methyltransferase activity, increased aberrant DNA methylation), whose activity decreases with aging. Further, aged mice fed high-polyamine chow demonstrated suppression of aberrant DNA methylation and a consequent increase in protein levels of lymphocyte function-associated antigen 1, which plays a pivotal role on inflammatory process. This review discusses the relation between polyamine metabolism and DNA methylation, as well as the biological mechanism of lifespan extension induced by increased polyamine intake.     

DNA photocleavage by porphyrin–polyamine conjugates

A series of polyamine–porphyrin conjugates bearing two (cis or trans position) or four units of spermidine or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by the means of UV–vis spectroscopy and we investigated their ability to cleave plasmid DNA in the presence of light. DNA binding and DNA photocleavage abilities were found to depend on structural characteristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet oxygen quencher (NaN₃) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen was the major species responsible for the cleavage of DNA previously observed. Collectively, these data show that polyamine–porphyrin conjugates could be promising phototherapeutic agents.     

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4'-diacetyldiphenylurea bis(guanylhydrazone) (DDUG)

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.