Strong patterns in homooligomer tracts occurrences in non-coding and in potential regulatory sites in eukaryotic genomes

Previous studies of the dinucleotides flanking both the 5' and 3' ends of homooligomer tracts have shown that some flanks are consistently preferred over others (1,2). In the first preferred group, the homooligomer tracts are flanked by the same nucleotide and/or the complementary nucleotides, e.g.,ATAn,TTAn,CCGn, where n = 2-5. Runs flanked by nucleotides with which they cannot base pair are distinctly disfavored. (In this group An/Tn are flanked by C and/or G; Gn/Cn are flanked by A/T, e.g.,CGAn,TnGG,GnAT). The frequencies of runs flanked by A or T, and G or C ("mixed"group) are as expected. Here we seek the origin of this effect and its relevance to protein-DNA interactions. Surprisingly, within the first group, runs flanked by their complements with a pyrimidine-purine junction (e.g.,TTAn,CnGG) are greatly preferred. The frequencies of their purine-pyrimidine junction mirror-images is just as expected. This effect, as well as additional ones enumerated below, is seen universally in eukaryotes and in prokaryotes, although it is stronger in the former. Detailed analysis of regulatory regions shows these strong trends, particularly in GC sequences. The potential relationship to DNA conformation and DNA-protein interaction is discussed.     

Distinct patterns in homooligomer tract sequence context in prokaryotic and eukaryotic DNA

The distributions of the junction sequences of homooligomer tracts of various lengths have been examined in prokaryotic DNA sequences and compared with those of eukaryotes. The general trends in the nearest and next to nearest neighbors to the tracts are similar for both groups. In both prokaryotes and eukaryotes A/T runs are preferentially flanked on either the 5' or the 3' ends by A and/or T. G/C runs are preferentially flanked by G and/or C. There is discrimination against A/T runs flanked by G or C and G/C runs flanked by A or T. However, whereas the distribution of prokaryotic homooligomer tract junction sequences was quite homogeneous, large variations were observed in the 5-fold larger eukaryotic database, increasing in magnitude from tracts of length 2 to 3 to 4 base pairs long. Possible DNA conformational implications and in particular DNA curvature and packaging aspects of prokaryotes and eukaryotes are discussed.     

The ordering of nucleotides in the DNA: strong pyrimidine-purine patterns near homooligomer tracts

Here, we study the frequencies of occurrence of homooligomers flanked by one base, XnU or UXn, where X = A, C, G, T and U not equal to X. Specifically, we search for preferences (or discriminations) in their nearest neighbor doublet, VV. Extensive analysis of the data base reveals striking patterns in such VVUXn or UXn VV oligomers (V = A, C, G, T). With very few exceptions, if the VV and Xn are composed of complementary nucleotides, those oligomers having a pyrimidine (Y)-purine (R) junction are preferred over those with an RY one. If the VV and Xn nucleotides are not complementary, the RY junction oligomers are preferred over their YR counterparts. These trends are observed consistently in eukaryotic and prokaryotic sequences. They are particularly striking in the YR greater than RY oligomers containing complementary nucleotides. The general preferences and discriminations described here are in the same direction as our previous results for homooligomer tracts. These recurrences, along with some additional universal "rules", aid in our understanding of the ordering of nucleotides in the DNA.     

Distinct patterns in the dinucleotide nearest neighbors to G/C and A/T oligomers in eukaryotic sequences

Summary The eukaryotic and prokaryotic databases are scanned for potential nearest-neighbor doublet preferences at the 5 and 3 flanks of some oligomers. Here we focus on oligomers containing alternating nucleotides, i.e., UV, UVUV, and UUVV where UV. Strong, consistent trends are observed in eukaryotic sequences. A/T alternation oligomers are preferentially flanked by A/T. G/C flanks are disfavored. G/C alternation oligomers are preferentially flanked by G/C. A/T flanks are disfavored. These trends are consistent with those observed previously for homooligomer tracts (Nussinov et al. 1989a,b). G/C tracts are preferentially flanked by G/C. A/T nearest neighbors are disfavored. The reverse holds for A/T tracts. Additional patterns are described here as well. The possible origin of these DNA composition and sequence trends is discussed. These trends are suggested to stem from protein-DNA interaction constraints.     

Sequence context of oligomer tracts in eukaryotic DNA: biological and conformational implications

Recent studies of homooligomer tracts suggest different characteristics from random sequence DNA (dA).(dT) and (dG).(dC) tracts are frequent in upstream regions and in some cases have been shown to be essential for regulation. Here we examine homooligomer occurrences in non-coding and coding eukaryotic sequences, focusing on the context in which the homooligomers occur. This analysis of sequences in the junction areas yields distinct and consistent characteristics. In particular, the nucleotide interrupting a run is most frequently complementary to the run. The base next to it is most frequently identical to the one constituting the run. For A or T runs the least frequent nearest and next to nearest neighbors are G or C. For G or C tracts the least frequent are A or T. Complementary oligomers behave similarly. These and additional trends are strongest for run lengths greater than or equal to 3. The computations are carried out on the whole eukaryotic database of greater than 4 x 10(6) nucleotides, separately for coding and non-coding regions. These same trends are evident for both groups, but are somewhat stronger for the non-coding regions. The context in which the homooligomers occur may yield some clues to DNA conformation and its biological implications.     

Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Dinucleoside(5′,5′) polyphosphates (Ap_nA, Ap_nG, Gp_nG, n=3–6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5′,5′) polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5′,5′) polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1–0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5′)-polyphospho-(5′)guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of Ap_nA, Ap_nG and Gp_nG (with n=3–6 in all cases). The reaction mixture was concentrated on a preparative C₁₈ reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.     

Interaction of mithramycin with isolated GC and CG sites

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)_n and A_n · T_n using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)_n appear to be bound better than those surrounded by A_n · T_n. Although no footprints are produced at T₉GCA₉ and T₁₅CGA₁₅, DNase I cleavage is enhanced with the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)_n · (CT)_n.     

The study of neighboring nucleotide composition and transition/transversion bias

Base substitution, the most common mutation thatone or more bases substitute another, is the main causethat creates individual variation, community diversityand the evolution of species. Studying the role andmechanism of base substitution could help peopl…     

Structural Basis of Stable Bending in DNA Containing An Tracts. Different Types of Bending

Abstract

Structural determinants of DNA bending of different types have been studied by theoretical conformational analysis of duplexes. Their terminal parts were fixed either in an ordinary low-energy B-like conformation or in “anomalous” conformations with a narrowed minor groove typical of A_n tracts. The anomalous conformations had different negative tilt angles (up to about zero), different propeller twists and minor groove widths. Calculations have been performed for DNA fragments A_nT_m, T_nA_m, A_nGCT_m, A_nCGT_m, T_mGCA_n, T_mCGA_n which are the models of the junction of two anomalous structures on A_n and T_m tracts. On the AT step of the A_nT_m fragment the minor groove can be easily narrowed so that a whole unbent fragment of anomalous structure is formed on A_n T_m. According to our energy estimates, there should not be any reliable bending on A_nT_m. In contrast, in all other cases there was a pronounced roll-like bending into the major groove in the chemical symmetry region. Calculations of the junction between the anomalous and ordinary B-like structure for G_nT_m and C_nA_m have shown that there is an equilibrium bending with a tilt component towards the chain having the anomalous structure at the 5′-end. From our calculations it is impossible to determine precisely the direction of bending, though it can be suggested that the roll component of bending might be directed towards the major groove. The anomalous structure is the main reason of bending; alternations of pyrimidines and purines can modulate the value and the direction of equilibrium bending (only the value in the case of self-complemantary fragments).

The results are consistent with the experimental data and promote a better understanding of the problem of DNA bending.     

Complex Analysis for Antiprotease–Protease Enzyme Gene Polymorphisms in Patients with Chronic Obstructive Pulmonary Diseases

A molecular genetic analysis of genes encoding the protease inhibitors ₁-antitrypsin (PI) and ₁-antichymotrypsin (AACT) was performed in patients with chronic obstructive pulmonary disease (COPD) (n = 239), cystic fibrosis (n = 57), and bronchiectasis (n = 33). In addition, the sample included children with chronic infant lung disease (n = 151) and nonobstructive chronic bronchitis (n = 34). Mutations Z and S of the ₁-antitrypsin gene (Glu342Lys and Glu264Val, respectively) resulting in severe enzyme deficiency and polymorphisms in the 3-untranslated region of the same gene (G1237A) and in the signal peptide for ₁-antichymotrypsin gene (Ala – 15Thr) were studied. No significant differences in the allele and genotype frequencies of these polymorphisms were revealed between the groups of patients and the control group. Promoter polymorphism G–1607GG in the interstitial collagenase gene (MMP1) was studied in patients with COPD, bronchiectasis, and chronic nonobstructive bronchitis. In COPD patients, the frequency of genotype GG/GG proved to be significantly higher than in the control group: 30.6 and 18.0%, respectively; ² = 6.58, p < 0.05; OR = 1.99 (95% CI 1.1–3.59). Thus, genetic polymorphism in the promoter of the MMP1 gene may be associated with an individual susceptibility to COPD.     

The study of neighboring nucleotide composition and transition/transversion bias

Base substitution is one of the raw fuels that produce genetic variation and drive evolution. Recent studies have shown that the genome components affect mutation patterns to some extent. In order to infer the correlation between the Transition/Transversion ratio (Ts/Tv) and the number of immediately adjacent A&T nucleotides, we investigated 3611007 Oryza sativa SNPs (including 45462 coding SNPs, and 242811 intronic SNPs) and 32019 Arabidopsis SNPs. The results show that Ts/Tv is negatively correlated with the number of immediately adjacent A&T in O. Sativa and Arabidopsis. We further calculated AT₂ (the number of SNPs whose immediately adjacent nucleotides are either A or T) and AT₀ (the number of SNPs whose immediately adjacent nucleotides are either C or G) for all 6 types of SNPs. C/G SNP of O. sativa and Arabidopsis has the highest AT₂/AT₀, which denotes C/G SNP may be influenced by the adjacent A&T nucleotides mostly. For SNPs in O. sativa, the neighboring effect of A&T nucleotides is limited to 2 nucleotides on both sides; for SNPs in Arabidopsis, the effect extends no more than 4 nucleotides on both sides.     

Generation of rules for expert systems by statistical methods of fermentation data analysis

Five new methods for determining the relations between kinetic data of fermentations are described and applied to an industrial antibotic fermentation process. The input data for these method are the elements of the distance matrix d_ij, which quantify the sum of the deviation squares between the time dependent kinetics x (t) of the fermentation runs i and j. For each measurable or calculable kinetic state variable, one n x n distance matrix must be calculated where n is the number of fermentation runs. All methods compare these distance matrices by statistical or graph-theoretical approaches. The algorithms obtained are universally applicable if enough kinetic data are available, especially from more than 10 comparable fermentation runs. The algorithms were developed for the use in knowledge acquisition modules of expert systems.     

Solution Structures of the Huntington's Disease DNA Triplets, (CAG)n

Summary

Highly polymorphic DNA triplet repeats, (CAG)_n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific PI enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)_n expansion. Nondenaturing gel electrophoresis and ID ¹H NMR studies of (CAG)₅ and (CAG)₆ reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0–2.0 mM that is typically required for 2D NMR experiments) both (CAG)₅ and (CAG)₆ exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)₅and (CAG)₆ are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG).,. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)₅ and (CAG)₆. We also studied a model duplex, (CGCAGCG)₂ that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2′-endo, anti) conformations, (ii) mismatched A?A base pairs are flanked by two Watson-Crick G?C base pairs and (iii) A?A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H—N1 hydrogen bond. The nature of A?A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)₅]₂ duplex where the adenines are ¹⁵N-labeled at N6. Temperature-and pH-dependent imino proton spectra, nondenaturing electrophoresis, and PI digestion data demonstrate that under a wide range of solution conditions longer (CAG)_n repeats (n>10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)₈₂₁ inserts in the Ml3 single-stranded DNA templates shows a replication bypass for the (CAG)₂₁ insert but not for the (CAG)₈ insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)., even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)_n may cause slippage during replication and thus may explain the observed length polymorphism.     

Coenzyme Q system in the classification of some ascosporogenous yeast genera in the families Saccharomycetaceae and Spermophthoraceae

The Co-Q systems of 11 strains representing the generaSchwanniomyces, Lodderomyces, Lipomyces, Nematospora andMetschnikowia were determined. All the genera were characterized by the Q-9 system except for the genusNematospora with needle-shaped ascospores. The only species,Nem. coryli, was found to have the Q-6 system. These results are discussed from the taxonomic point of view. This constitutes Part VII of a series entitled “Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms.” The abbreviations used here for coenzyme Q or ubiquinone are: Co-Q, coenzyme Q; Co-Q _n , Q-n or Q _n withn denoting a specified number of isoprene units in a side chain, e.g., Co-Q₆, Q-6, Q₆, etc.     

Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains

We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing the protein with the capacity to be allosterically regulated by adenine nucleotides (J?msen, J., Tuominen, H., Salminen, A., Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327–333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated by diadenosine polyphosphates (AP _n A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2′(3′)-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding. The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it to more rapidly undergo conformational changes in response to binding.     

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (g _j ), we compared the effects of 2mm 1-heptanol and 20 μm 2-(methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A₂C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (r_ss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6±1.1% (mean ±sem,n=5) and 9.8±0.6% (n=5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2mm heptanol uncoupled cell pairs completely (n=6), whereas 20 μm A₂C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n=5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased r_ss by 14.9±3.0% (n=5), i.e., decreased cholesterol domain fluidity, whereas A₂C had no effect on r_ss (−0.4±6.7%,n=5). Following an increase of cellular “cholesterol” content (by loading the cells with DHE), 2mm heptanol did not uncouple cell pairs completely:g _j decreased by 80±20% (range 41–95%,n=5). The decrease ing _j was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of non-junctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreasesg _j by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.     

Chain length characterization of oligodeoxyribonucleotides by analytical ultracentrifugation

Analytical ultracentrifugation was used to determine chain length of oligomers in the deoxyribonucleotide series. Uncertainties with respect to partial specific volume of the solute and questions of charge compel a semiempirical approach: variations of these parameters introduce fluctuations well within the experimental error of the method. Measurements of the sedimentation equilibria for three homooligomer series—d(pT)_n, d(pA)_n and d(pC)_n—as well as a number of oligomers of varying base composition generated straight-line plots passing through the origin.     

Friedreich's ataxia (GAA)n•(TTC)n repeats strongly stimulate mitotic crossovers in Saccharomyces cerevisae

Expansions of trinucleotide GAA•TTC tracts are associated with the human disease Friedreich''s ataxia, and long GAA•TTC tracts elevate genome instability in yeast. We show that tracts of (GAA)₂₃₀•(TTC)₂₃₀ stimulate mitotic crossovers in yeast about 10,000-fold relative to a “normal” DNA sequence; (GAA)_n•(TTC)_n tracts, however, do not significantly elevate meiotic recombination. Most of the mitotic crossovers are associated with a region of non-reciprocal transfer of information (gene conversion). The major class of recombination events stimulated by (GAA)_n•(TTC)_n tracts is a tract-associated double-strand break (DSB) that occurs in unreplicated chromosomes, likely in G1 of the cell cycle. These findings indicate that (GAA)_n•(TTC)_n tracts can be a potent source of loss of heterozygosity in yeast.