Protonated polynucleotide structures. 18. Interaction of oligocytidylates with poly (G).

The faculty for and degree of oligo(C)-poly(G) interaction is described as an essentially chain length - sensitive phenomenon. At neutral pH under suitable experimental conditions, oligocytidylates of chain length greater than four associate with poly(G) to form double-stranded structures, as does poly(C). The extent of complex formation increases with degree of polymerization. The complex at acid pH is shown to be triple-stranded, of stoicheometry 2C/1G. The observation of a 2G/1C artifact is discussed.     

Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

Cooperative nonenzymic base recognition--a study of interaction of oligoguanylic acid with poly C

The interaction between poly C and (Gp)_nG(n = 1,2) in dilute solution was investigated spectrophotometrically in 0.1M phosphate buffer pH 7.2 under conditions unfavorable for the formation of self-associated complexes of oligoguanylic acids. Two isosbestic points were observed when poly C was titrated gradually with GpGpG, one at 232–233 mμ(in the range of 0–33% poly C) and one around 238 mμ (in the range of 50–100% poly C). The melting temperature (T_m) of the 1:1 poly C: (Gp)nG complexes (n = 1,2) of varying concentration were determined. The equilibrium properties of the 1:1 complexes can be described by two interaction parameters, namely, (i) cooperative stacking interaction between the first nearest neighbor of the adsorbed oligomer, and (ii) intrinsic association constant of the adsorbed oligomer with its polymeric site, since the cooperative helix–coil transition particularly in the smaller oligonucleotide can be described by an “all or none” model. Based on such a model the enthalpy of stacking inteaction-dependent T_m values yielded directly the sum of the enthalpy of stacking interaction and of basepairing (which is dependent on the chain length of the oligomer) and the value of S, the stability constant of a G–C pair within a helix. The enthalpy of formation of G–C pair is then calculated as ?6.3 kcal/base pair either from the chain length dependent enthalpy term or from the temperature coefficient of S values. From the S value and the association constant of 1:1 GpGpGpC:GpCpCpC complex, other thermodynamic parameters such as nucleation parameter (β) and free energy of stacking interaction can be obtained.     

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.     

Complex formation between ribosomal protein S1, oligo-and polynucleotides: chain length dependence and base specificity.

In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The complex formation is Mg2+ dependent at low salt concentrations and becomes Mg2+ independent at an ionic strength greater than 90 mM. Oligouridylates of increasing chain length reach an optimal KA of 3-3-10(7) M-1 at a chain length of n=13-14. Protein S1 contains one binding site for long chain oligouridylates, such as U12, and the standard-free-energy change on binding caused by one Pu increment is 0.41 kcal/mol, when n varies between five and fourteen. Complex formation is insensitive to the capacity of the homopolynucleotide bases to form hydrogen bonds. Homopolynuceotides, however, showing a Tm less than 250 in the buffer system used show an increased affinity for S1 compared to poly(A) and poly(C) (Tm greater than 40 degrees). The data are discussed with respect to the proposed binding of protein S1 to the 3-terminal end of the 16S RNA.     

Interaction of ethidium bromide with synthetic double-stranded polyribonucleotides

The interaction of ethidium bromide (EtBr) with double helical synthetic polyribonucleotides poly(G).poly(C), poly(A).poly(U) and poly(I).poly(C) has been investigated by the method of isothermal microcalorimetry and according to the character of changes on the spectra of circular dichroism, absorption and fluorescence at binding. The calculations showed that at binding of EtBr with poly(A).poly(U) the saturation stechiometry was one EtBr molecule per 2 base pairs with binding constant (2.5 +/- 0.5).10(6) M-1 at 30 degrees C and 0.1 M. NaCl. In the case of binding of EtBr with poly(G).poly(C) and poly(I).poly(C) the saturation stechiometry was not less than 0.2 EtBr molecule per 1 base pair with binding constant (4 +/- 1).10(3) M-1 and (1.5 +/- 0.3).10(4) M-1 respectively, at 25 degrees C and 0.1 M NaCl. The binding enthalpies of EtBr with poly(A).poly(U) and poly(G).poly(C) have been determined to be (-7.5 +/- 0.5) Kcal per 1 mol of bound EtBr in average for both polymers. It has been shown that the observed strong selectivity of EtBr binding with polyribonucleotides is of entropic origin.     

Affinity separation of polyribonucleotide-binding human blood proteins

Using affinity columns with immobilized poly(A), poly(G), poly(U), poly(C), and poly(A).poly(U) and poly(G) x poly(C) duplexes several polyribonucleotide-binding blood plasma proteins have been captured. Albumin and keratins K1 and K2e have been detected to bind polypurine tracts. The in vitro glycated albumin binds poly(A) and poly(G) more efficiently than the unmodified protein. The major polypyrimidine-binding blood plasma protein (28 kDa) can catalyze the hydrolysis of poly(U).     

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the poly G-poly C complex by incorporation of adenosine in the purine chain

Antiviral and interferonogenic activity of the complexes of poly(G,A) . poly(C) and poly(G) . poly(C) was studied in mice and cell cultures. Three out of 4 complexes of poly(G,A) . poly(C) had insignificant antiviral and interferonogenic activity in chick embryo cells. One of the complexes induced low levels of interferon production in mice and decreased the rate of their death from experimental forest-spring encephalitis. The activity of poly(G) . poly(C) in the above cell systems was much more pronounced. Unlike this complex, some complexes of poly(G,A) . poly(C) showed a noticeable activity in the cells of Primates. The effect of the noncomplementary base in the purine thread of poly(G) . poly(C) on its biological activity and nucleotide composition is discussed.     

New wrinkles on polynucleotide duplexes

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As4T):poly d(As4T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c = 7.40nm, gamma = 90 degrees. The c dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4(3) helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain C3'-endo furanose rings and the pyrimidine nucleotides C2'-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

Repair of Single- and Multiple-Substitution Mismatches during Recombination in Streptococcus Pneumoniae

The use as genetic markers, during transformation of Streptococcus pneumoniae, of 19 sequences differing from wild type, located throughout the amiA locus, enabled us to examine the fate of 24 single- and 11 multiple-mismatches during recombination. Tentative mismatch ranking as a function of decreasing repair efficiency by the Hex mismatch repair system is G/T = A/C = G/G (maximum repair: 90-95%) greater than C/T (mostly 75 to 90% repair) greater than A/A (from 50 to 90% repair) greater than T/T (50-65% repair) greater than A/G (from 0 to 20% repair) greater than C/C. No indication of correction of the latter has been obtained. Over the limited number of samples examined, we observed no influence of the base composition of the surrounding sequence on correction efficiency for both transition mismatches and for G/G and C/C. Variations in the surrounding sequence affect repair of A/G and C/T, and, even more strongly, of A/A and T/T. No simple correlation to the G:C content of the surrounding sequence is apparent from our results, in contrast to the conclusion drawn for the Mut mismatch repair system of Escherichia coli. Examination of the fate of multiple mismatches suggests that C/C may sometimes impede recognition of otherwise corrected mismatches.     

鲟源病原性嗜水气单胞菌拮抗芽孢杆菌的鉴定及其生物学特性

从养殖池污泥中分离筛选了1株优良的鲟源嗜水气单胞菌拮抗芽孢杆菌G1,其对鲟源嗜水气单胞菌S1产生的抑菌圈直径为18.50 mm。通过API50CH细菌鉴定系统以及16S rRNA序列分析法,菌株G1被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),GenBank登录号HM245965.1,其16S rRNA序列与基因库中芽孢杆菌属菌株的16S rRNA序列有99%100%的同源性,而且与解淀粉芽孢杆菌Ba-74501(GenBank登录号:DQ422953.1)的亲缘关系最近。菌株G1的最适生长pH值为7,最适生长温度为30°C,其在30°C、200 r/min条件下的生长曲线为:0 6 h为生长延迟期,6 54 h为对数生长期,54 90 h为稳定期,90 h以后为衰亡期。此外,菌株G1对其他实验选用的病原性嗜水气单胞菌也表现出良好的拮抗活性。本实验结果有利于填补嗜水气单胞菌拮抗菌在分类地位、生物学特性等方面的不足,为鲟鱼嗜水气单胞菌病的生物防控提供科学资料。     

Raman spectroscopic measurements in self‐pressurized aqueous solutions above 100°C: The melting of poly(G) and poly(G) · poly(C)

We have studied by Raman spectroscopy the thermal behavior of associated polyguanylic acid [poly(G)] and polyguanylic–polycytidylic acid [poly(G) · poly(C)] in self‐pressurized aqueous solutions contained in sealed capillary tubes. The associated polynucleotides were found to be very resistant to heat, but evidence of thermal degradation was observed after melting of the helical structures. The cooperative melting transition of the four‐stranded complex of poly(G) was located at 141°C in 0.5M KCl, 135°C in 0.5M NaCl, 129°C in 0.5M LiCl, 123°C in 0.1M tetramethylammonium perchlorate, and 105°C in 0.1M tetraethylammonium bromide solutions. The transition was observed at 130°C in poly(G) · poly(C) (in 0.5M NaCl). The results in this case show that a four‐stranded poly(G) complex is formed following the melting of the double helix. © 1999 John Wiley & Sons, Inc. Biopoly 49: 21–28, 1999