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PubMed2003

Computational methods and evaluation of RNA stabilization reagents for genome-wide expression studies.

Gene expression studies require high quality messenger RNA (mRNA) in addition to other factors such as efficient primers and labeling reagents. To prevent RNA degradation and to improve the quality of gene array expression data, several commercial reagents have become available. We examined a conventional hot-phenol lysis method and RNA stabilization reagents, and generated comparative gene expression profiles from Escherichia coli cells grown on minimal medium. Our data indicate that certain RNA stabilization reagents induce stress responses and proper caution must be exercised during their use. We observed that the laboratory reagent (phenol/EtOH, 5:95, v/v) worked efficiently in isolating high quality mRNA and reproducibility was such that reliable gene expression profiles were generated. To assist in the analysis of gene expression data, we wrote a number of macros that use the most recent gene annotation and process data in accordance with gene function. Scripts were also written to examine the occurrence of artifacts, based on GC content, length of the individual open reading frame (ORF), its distribution on plus and minus DNA strands, and the distance from the replication origin.

Base Composition↗

PubMed2002

A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases.

We have developed an assay for online detection of DNA cleavage by restriction endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured K(M) and k(cat) values for the restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many restriction endonuclease variants in parallel.

Base Composition↗

PubMed1987

NMR studies of the stable mismatch purine-thymine in the self-complementary d(CGPuAATTTCG) duplex in solution.

One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.

Base Composition↗

PubMed2006

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker.

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Base Sequence↗

PubMed1983

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.

Adenine↗

PubMed1984

Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.

The amino-terminal domain of the phage 434 repressor forms cocrystals with a synthetic phage 434 operator. The cocrystals diffract to at least 4 A, and x-ray crystallographic analysis of them is in progress. An analysis of the packing in the cocrystals shows that complexes consisting of dimers of amino-terminal domain bound specifically to operators are stacked end to end in longer protein-DNA rods parallel to the unit cell body diagonals. The DNA in the complexes has 10.5 base pairs per turn and a rise per base of 3.26 A--values consistent with B-form DNA--indicating that DNA is neither unwound nor overwound by bound repressor. The packing analysis suggests an approach that might facilitate the cocrystallization of other DNA-binding proteins with the DNA they recognize.

Base Composition↗

PubMed1989

Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme.

A shortened form of the intervening sequence of the self-splicing RNA from Tetrahymena thermophila catalyzes sequence-specific cleavage of RNA. Cleavage site selection involves a base-pairing interaction between the substrate RNA and a binding site within the intervening sequence. Single-base changes in this binding site were previously shown to alter substrate specificity in a predictable manner. To examine the generality with which substrate specificity can be altered, six variant catalytic RNAs (ribozymes) have been produced with two- or three-base changes in the active site. Each ribozyme cleaves its predicted substrate. The conditions required for good reactivity and for discrimination against cleavage at mismatched sites vary and were independently determined for each ribozyme.

Animals↗

PubMed1982

DNA fragment conformations IV - Helix-coil transition and conformation of d-CCATGG in aqueous solution by 1H-NMR spectroscopy.

The helix-coil transition and conformation of d-CCATGG were investigated using 1H-NMR spectroscopy at various frequencies (90, 276, 400 MHz). The changes in the chemical shifts and linewidths of imino protons between 5 degrees and 35 degrees C show that the d-CCATGG fraying process consists of two stages: the external dC.dG base pairs open at first, th internal dC.dG and central dA.dT base pairs then open simultaneously at higher temperatures similar to the case of d-ACATGT. The midpoint temperatures, the helix and coil proportions and the dissociation constant were determined from the sigma = f(t degree) curves of the base and sugar protons. The results indicate that the midpoint temperature increases with the number of the dG.dC base pair in a given size sequence, while the dissociation enthalpy appears to be independent. The difference between the T1 value of a base proton of the external and internal residues of the same nature is found to be a good criterion for base proton assignment. The high predominance of the S conformation for all residues shows that d-CCATGG duplexes adopt the B-helical conformation.

Base Composition↗

PubMed1983

Complete nucleotide sequence of the human corticotropin-beta-lipotropin precursor gene.

The nucleotide sequence of an 8658-base-pair human genomic DNA segment containing the entire corticotropin-beta-lipotropin precursor gene has been determined, and some sequence features of the gene and its flanking regions have been analysed. The gene is composed of 7665 base pairs including two introns of 3708 and 2886 base pairs. Comparison of the 5'-flanking sequences of the human, bovine and mouse corticotropin-beta-lipotropin precursor genes reveals the presence of a highly conserved region, which contains sequences of 14-15 base pairs homologous with sequences located upstream of the mRNA start site of other glucocorticoid-regulated genes.

Adrenocorticotropic Hormone↗

PubMed1993

The crystal structure of N4-methylcytosine.guanosine base-pairs in the synthetic hexanucleotide d(CGCGm4CG).

The structure of d(CGCGm4CG) were m4C = N4-methylcytosine has been determined by crystallographic methods. The crystals are multifaced prisms, with orthorhombic space group P2(1)2(1)2(1) and unit cell dimensions of a = 17.98, b = 30.77 and c = 44.75A. The asymmetric unit consists of one duplex of hexanucleotide and 49 waters. The R-factor is 0.189 for 1495 reflections with F > or = sigma(F) to a resolution limit of 1.8A. The double helix has a Z-DNA type structure which appears to be intermediate in structure to the two previously characterised structure types for Z-DNA hexamers. The two m4C.G base-pairs adopt structures that are very similar to those of the equivalent base-pairs in the structure of the native sequence d(CGCGCG) except for the presence of the methyl groups which are trans to the N3 atoms of their parent nucleotides and protrude into the solvent region. The introduction of the modified base-pairs into the d(CGCGCG) duplex appears to have a minimal effect on the overall base-pair morphology of the Z-DNA duplex.

Base Composition↗

PubMed1991

Studies on 1,3-diaryltriazene analogues of berenil: molecules with potential GC base-pair selectivity.

1,3-Bis(4-acetylphenyl)triazene (II): C16H15N3O2, Mr = 281.32, monoclinic, P2(1)/c, a = 14.002 (5), b = 12.359 (3), c = 8.457 (3) A, beta = 96.35 (2) degrees, V = 1454.5 A3, Z = 4, Dx = 1.287 Mg m-3, Cu K alpha, lambda = 1.54178 A, mu = 0.672 mm-1, F(000) = 592, T = 294 K, final R = 0.089 for 1890 unique observed reflections. 1,3-Bis(4-[2-dimethylamino)ethoxycarbonyl]phenyl)triazene monohydrate (III): C22H29N5O4.H2O, Mr = 445.52, triclinic, P1, a = 9.500 (2), b = 11.753 (3), c = 13.328 (2) A, alpha = 62.84 (1), beta = 66.60 (2), gamma = 77.58 (2) degrees, V = 1214.1 A3, Z = 2, Dx = 1.172 Mg m-3, Cu K alpha, lambda = 1.54178 A, mu = 0.640 mm-1, F(000) = 476, T = 294 K, final R = 0.063 for 1100 unique observed reflections. Both crystal structures have extended conformations for the 1,3-diaryltriazene groups, with a cisoid arrangement of phenyl rings. The terminal N--N bonds of each triazene are non-equivalent, and a hydrogen atom has been located in the N==N--NH moiety. Extensive molecular-orbital (MNDO) calculations on the model core 1,3-diphenyltriazene system have confirmed that this geometry is energetically favoured, and have revealed the shape of the energy surface for rotation about the N--NH bond in the triazene linkage.

Base Composition↗

PubMed1995

Sequence specificity of tetrahydrobenzopsoralen photobinding to DNA.

The sequence specificity of photobinding to DNA of two tetrahydrobenzopsoralen derivatives has been investigated by testing the photoreactivity toward a number of self-complementary oligonucleotides. The thermodynamic constant for noncovalent binding to each DNA sequence was evaluated. The extent of photoreactivity was greatly dependent upon base composition. The two tetracyclic compounds show similar behavior in comparison to other bifunctional derivatives. Their overall rate constants were greatly enhanced in comparison to classical psoralens. However, their high efficiency of covalent binding is counterbalanced by low affinity for noncovalent interaction with DNA.

Adenine↗

PubMed2004

Selective and mutational patterns associated with gene expression in humans: influences on synonymous composition and intron presence.

We report the results of a comprehensive study of the influence of gene expression on synonymous codons, amino acid composition, and intron presence and size in human protein-coding genes. First, in addition to a strong effect of isochores, we have detected the influence of transcription-associated mutational biases (TAMB) on gene composition. Genes expressed in different tissues show diverse degrees of TAMB, with genes expressed in testis showing the greatest influence. Second, the study of tissues with no evidence of TAMB reveals a consistent set of optimal synonymous codons favored in highly expressed genes. This result exposes the consequences of natural selection on synonymous composition to increase efficiency of translation in the human lineage. Third, overall amino acid composition of proteins closely resembles tRNA abundance but there is no difference in amino acid composition in differentially expressed genes. Fourth, there is a negative relationship between expression and CDS length. Significantly, this is observed only among genes with introns, suggesting that the cause for this relationship in humans cannot be associated only with costs of amino acid biosynthesis. Fifth, we show that broadly and highly expressed genes have more, although shorter, introns. The selective advantage for having more introns in highly expressed genes is likely counterbalanced by containment of transcriptional costs and a minimum exon size for proper splicing.

Amino Acids↗

PubMedDate not supplied

[Study of the flexibility of complementary dinucleoside phosphates by the Monte-Carlo method].

Monte-Carlo calculations have revealed a significant flexibility of the double stranded dinucleoside phosphates AA: TT, AT: AT, TA: TA, GG: CC, GC: GC and CG: CG within the limits of the B-family of forms. Fluctuations of the dihedral angles and of the phase angle of pseudorotation, P, have been found to be 5-20 degrees and 10-20 degrees respectively. The r.m.s. deviation of the helical winding angle varies from 3 degrees to 6 degrees for different dimers. The G: C-containing dimers proved to be more rigid that the A: T-containing ones, though the methyl groups of thymines take part in stabilization of AA: TT and AT: AT dimers. The propeller angles in A: T and G: C pairs are of opposite sing. The pyrimidine-(3'-5')-purine dinucleoside phosphates demonstrate the least stability. All the dimers proved to be more flexible when opened into both grooves than in perpendicular directions. Due to the greater value of the glycosidic angle in the 3'-monomer the base pairs are opened into the wide groove. A connection between opening of the base pairs and bending of the double helix for different nucleotide sequences has been discussed. This might be important for the DNA package in nucleosomes.

Base Composition↗

PubMed1992

The application of Markov chain analysis to oligonucleotide frequency prediction and physical mapping of Drosophila melanogaster.

Here we compare several methods for predicting oligonucleotide frequencies in 691 kb of Drosophila melanogaster DNA. As in previous work on Escherichia coli and Saccharomyces cerevisiae, a relatively simple equation based on tetranucleotide frequencies can be used in predicting frequencies of higher order oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.07 with a sample standard deviation of .55. This simple predictor arises by considering each base on the sense strand of D. melanogaster to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate than the random predictor. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in selecting probes to fingerprint clones in contig mapping. Once again, this equation well predicts the occurrence of higher order oligonucleotides, supporting our hypothesis that this predictor holds in evolutionarily diverse organisms. When ranked from highest to lowest abundance, the observed frequencies of oligomers of a given length are closely tracked by the predicted abundances of a 3rd order Markov chain. Through use of the dependence of oligomer frequencies on base composition, we report a list of oligomers that will be useful for the completion of a cosmid physical map of D. melanogaster. Presently, the library is such that it will be possible to construct large contigs using only 30 oligonucleotide probes to fingerprint cosmids.

Animals↗

PubMed1992

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. 3. Incorporation of adenosine and uridine residues.

We have used [32P]-labeled hairpin oligonucleotides to study template-directed synthesis on templates containing one or more A or T residues within a run of C residues. When nucleoside-5'-phosphoro(2-methyl)imidazolides are used as substrates, isolated A and T residues function efficiently in facilitating the incorporation of U and A, respectively. The reactions are regiospecific, producing mainly 3'-5'-phosphodiester bonds. Pairs of consecutive non-C residues are copied much less efficiently. Limited synthesis of CA and AC sequences on templates containing TG and GT sequences was observed along with some synthesis of the AA sequences on templates containing TT sequences. The other dimer sequences investigated, AA, AG, GA, TA, and AT, could not be copied. If A is absent from the reaction mixture, misincorporation of G residues is a significant reaction on templates containing an isolated T residue or two consecutive T residues. However, if both A and G are present, A is incorporated to a much greater extent than G. We believe that wobble-pairing between T and G is responsible for misincorporation when only G is present.

Adenine↗

PubMed1997

Study of RNA structure by pyrene-labeled oligonucleotides.

Fluorescent-labeled oligonucleotides containing 2'-(1-pyrenylmethyl) uridine (Upy) that have G or C adjacent to 3'-site of Upy, I and II, respectively, showed a remarkable increase in the emission intensity when they hybridized with the complementary oligoribonucleotides (ORN). However such change was not observed when they hybridized with the complementary oligodeoxyribonucleotides(ODN). Tm values derived from the UV melting curves for I-ORN and II-ORN duplexes were 5 degrees C and 10 degrees C lower than the normal duplexes without Upy, respectively. The intensity of induced CD based on the pyrene groups for the I-ORN and II-ORN duplexes were a half of that for the corresponding I-ODN and II-ODN duplexes. These results suggest that pyrene groups were released from the stacking interaction upon duplex formation and that the remarkable change of emission intensity was then occurred. Based on these properties, it is suggested that Upy-probes that have G or C adjacent to 3'-site of Upy can be useful to search the single strand region of RNA.

Base Composition↗

PubMed1994

Synthesis and properties of oligodeoxyribonucleotide-polyethylene glycol conjugates.

Pools of oligonucleotide conjugates consisting of 10-400 different molecular species were synthesized. The conjugates contained a varying number of ethylene glycol units attached to 3'-terminal, 5'-terminal and internal positions of the oligonucleotides. Conjugate synthesis was performed by phosphoramidite solid phase chemistry using suitably protected polyethylene glycol phosphoramidites and PEG-derivatized solid supports containing polydisperse PEGs of various molecular weight ranges. The pools were analyzed and fractionated by chromatographic and electrophoretic techniques, and the composition of isolated conjugates was revealed by matrix-assisted laser desorption/ionization mass spectrometry. The number and attachment sites of coupled ethylene glycol units greatly influence the hydrophobicity of the conjugates, as well as their electrophoretic mobilities. Conjugation had little effect on the hybridization behavior of oligonucleotide conjugates with unmodified complementary oligonucleotide strands. Melting temperatures were between 67 and 73 degrees C, depending on the size and number of coupled PEG chains, compared to 68 degrees C for the unmodified duplex. Conjugates with PEG coupled to both 3'- and 5'-terminal positions showed a more than 10-fold increase in exonuclease stability.

Base Sequence↗