Reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate. Nuclear magnetic resonance studies of the corresponding S-peptide

The n.m.r. spectra of native S-peptide and of S-peptide II, a derivative obtained after reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate, both in D2O and in urea-d4, were obtained with a 270 MHz Fourier transform spectrometer. From these spectra it was possible to assign most of the proton resonances of the peptide and the position of the labelling group, the alpha-NH2 of Lys-1, was also deduced.     

1H-n.m.r. study of the folding of ribonuclease 12-(beta-(3-pyridyl)-L-Ala) S-peptide (1-14)

The 1H-n.m.r. spectra (360 MHz) of 12-(beta-(3-pyridyl)-L-Ala) ribonuclease S-peptide (1-14), a tetradecapeptide incorporating (beta-3-pyridyl-L-Ala) instead of His at position 12, have been assigned. The shift vs. temperature dependence has been analyzed at three different pD's in terms of a two-state helix (3-13) in equilibrium coil equilibrium, and the corresponding values for the thermodynamic quantities delta H degrees and delta S degrees determined. Helix populations at 0 degrees C have been measured as a function of pD, showing their dependence on two apparent pKa's at approximately 3.3 and 5.5, with a maximum at pD approximately 4.2. All the obtained results show that the new peptide has very similar folding properties to those shown by S-peptide and particularly to those of C-peptide. The 3-13 helix formed is stabilized by two interactions: a salt-bridge Glu 2-...Arg 10+ and a partial stacking between the aromatic rings of residues Phe 8 and His 12. Calculations involving ring current shifts and potential energies validate the possible existence of this latter interaction, which must present a local geometry defined by chi 81 180 degrees, chi 82 100 degrees, chi 121-60 and chi 122 80.     

The 10 kb Drosophila virilis 28S rDNA intervening sequence is flanked by a direct repeat of 14 base pairs of coding sequence

Most repeat units of rDNA in Drosophila virilis are interrupted in the 28S rRNA coding region by an intervening sequence about 10 kb in length; uninterrupted repeats have a length of about 11 kb. We have sequenced the coding/intervening sequence junctions and flanking regions in two independent clones of interrupted rDNA, and the corresponding 28S rRNA coding region in a clone of uninterrupted rDNA. The intervening sequence is terminated at both ends by a direct repeat of a fourteen nucleotide sequence that is present once in the corresponding region of an intact gene. This is a phenomenon associated with transposable elements in other eukaryotes and in prokaryotes, and the Drosophila rDNA intervening sequence is discussed in this context. We have compared more than 200 nucleotides of the D. virilis 28S rRNA gene with sequences of homologous regions of rDNA in Tetrahymena pigmentosa (Wild and Sommer, 1980) and Xenopus laevis (Gourse and Gerbi, 1980): There is 93% sequence homology among the diverse species, so that the rDNA region in question (about two-thirds of the way into the 28S rRNA coding sequence) has been very highly conserved in eukaryote evolution. The intervening sequence in T. pigmentosa is at a site 79 nucleotides upstream from the insertion site of the Drosophila intervening sequence.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A.

1H NMR spectroscopy at 100 MHz was used to determine the first-order rate constants for the 1H-2H exchange of the H-2 histidine resonances of RNase-A in 2H2O at 35 degrees C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2H2O at 35 degrees C and pH meter reading 11 caused irreversible denaturation of RN-ase-A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1H-3H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [J. Biochem, (Tokyo) 75, 1197-1200 (1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first-order rate constant for the exchange of His-48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5 The corresponding values for His-119 show a decrease and those for His-12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase-A (probably due to the titration of Tyr-25) which allows His-48 to become accessible to solvent. 1H NMR spectra of S-protein and S-peptide, and of material partially deuterated at the C-2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase-A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun., 936-937]. The chemical shifts of the C-2 and C-4 protons of histidine-12 of S-peptide are followed as a function of pH and a pK' value of 6.75 is obtained. The reassignment of the three C-2 histidine resonances of S-protein is confirmed by partial deuteration studies. The pK' values obtained from titration of the H-2 resonances of His-48, His-105 and His-119 are 5.3, 6.5 and 6.0, respectively. The S-protein is less stable to acid than RNase-A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26 degrees C. His-48 in S-protein titrates normally and has a lower pK than in RN-ase-A probably because of the absence of Asp-14, which in RN-ase-A forms a a hydrogen bond with His-48 and causes it to be inaccessible to solvent, at pH values below 9.     

Construction of a dense SNP map for bovine chromosome 6 to assist the assembly of the bovine genome sequence

A linkage map was constructed for bovine chromosome 6 (BTA6), using 399 single nucleotide polymorphisms (SNPs) detected primarily from PCR-resequencing. The efficiency of SNP detection was highly dependent on the source of sequence information chosen for primer design (BAC-end sequences, introns or promoters). The SNPs were used to build a linkage map comprising 104 cM on BTA6. The SNP order in the linkage map corresponded very well with radiation hybrid (RH) maps available for BTA6 as well as with expected positions in the human comparative map, but diverged significantly from the current assembly of the bovine genome (Btau_3.1). When performing linkage analysis with the marker order suggested from the Btau_3.1 we observed an expansion of the genetic map from 104 cM to 137 cM, strongly suggesting a reordering of scaffolds in the current version of the bovine genome assembly. The extent of LD on BTA6 was evaluated by calculating the average r ² for SNP pairs separated by given distances. The decline of LD was rapid with distance, such that r ² was 0.1 at 100 kb. Our results indicate that linkage mapping will be a valuable source of information for correcting errors in the current bovine assembly. These errors were sufficiently frequent to be of concern for the accuracy of mapping QTL with panels of SNPs whose positions are based on the current assembly.     

Modification of bovine pancreatic ribonuclease A with the site-specific reagent 4-arsono-2-nitrofluorobenzene. Spectrophotometric titration of arsononitrophenyl ribonuclease A derivatives

The 4-arsono-2-nitrophenyl chromophore can serve as a versatile spectrophotometric probe of the surface structure of proteins. Values of pK1' and pK2' for the arsonic acid ionizations are near 3 and 8, respectively, and the presence of nearby positive and negative charges produces substantial alterations in the spectral response of the probe. Changes in the extinction at the wavelength of maximum difference are 30-50% of the extinction coefficients, epsilonmax, for each ionization of the arsonic acid moiety. The titration of 41-(4-arsono-2-nitrophenyl)ribonuclease A indicates that the arsonate dianion binds near the active-site histidine residues. With protonation of a carboxylate side chain in the acidic region, presumably aspartic acid-121, the active site is disrupted. The 41-(4-arsono-2-nitrophenyl) group interacts to a greater degree with the histidine-119 side chain than it does with the histidine-12 residue. Interactions of uridine or 3'-cytidylic acid with the ligand-binding region of 41-(4-arsono-2-nitrophenyl) ribonuclease A modify the spectrophotometric response extensively. 3'-Cytidylic acid binds 41-(4-arsono-2-nitrophenyl) ribonuclease A with an affinity 300 times less than that for native ribonuclease A and 17 times lower than that for 41-(2,4-dinitrophenyl) ribonuclease A. The arsononitrophenyl chromophore is responsive to changes in the active site of ribonuclease A induced by such perturbants as ligand binding, chemical modification, and both acid and thermal denaturation.     

A crystalline A14-(2-nitro-4-trimethylammoniophenyl) derivative of bovine insulin

[O-(2-Nitro-4-trimethylammoniophenyl)-TyrA 14]insulin (bovine) is a product formed on reaction of bovine insulin with the hydrophilic reagent 1-fluoro-2-nitro-4-trimethyl-ammoniobenzene iodide (TAN-F) in an aqueous buffer at pH 8.00. The derivative was isolated and its purity established by standard procedures. The identity of the derivative was determined by degrative studies with alpha-chymotrypsin. The addition of zinc to the above reaction decreases the yield of the title derivative, but increases the yield of the [N alpha-TAN-GlyA1] derivative. [N alpha-Boc-GlyA1]insulin was also reacted with the above mentioned reagent in an attempt to improve the yield of the A14-tyrosine derivative. The biological activity of this microcrystalline derivative was found to be 12.4 units/mg as measured by the mouse convulsion assay.     

Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.     

Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA

Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.     

A theoretical investigation on the sequence selective binding of mitoxantrone to double-stranded tetranucleotides.

Theoretical computations are performed on the comparative binding energetics of mitoxantrone (MX), a newly synthesized intercalating anthraquinone antitumor drug, to six representative double-stranded tetranucleotides: d(GCGC)2, d(CGCG)2, d(ATAT)2, d(TATA)2, d(GTGT), d(ACAC), and d(CCGG)2. The computations are performed with the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The best binding configuration of mitoxantrone locates its two side chains in the major groove. A considerable preference is elicited for intercalation of the chromophore ring in a pyrimidine (3'-5') purine sequence rather than the isomeric purine (3'-5') pyrimidine sequence. Contrary to the situation encountered with "simple" intercalators, in which this preference is generally attributed solely to differences in the energies of unstacking necessary to generate the intercalation sites, the preference is dictated in MX to a large extent by the intermolecular interaction energy term. This result is imposed by the interactions of the side chains of MX with the oligonucleotide.     

A theoretical investigation on the sequence selective binding of adriamycin to double-stranded polynucleotides.

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of adriamycin (ADM) to five self-complementary double-stranded hexanucleotides. Among the two regularly alternating hexanucleotides d (TATATA)2 and d (CGCGCG)2, a stronger binding is predicted for the former. The strongest complex is computed, however, for the mixed hexanucleotide d (CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. The overall sequence preference is the result of an intricate interplay of sequence preferences of the constituents in particular of daunosamine and the 9-OH substituent. Altogether, the selective base pair recognition by adriamycin cannot be defined in terms of the two base pairs implicated in the intercalation site alone but must be expressed in terms of a triplet of base pairs.     

A search for base analogs to enhance third-strand binding to 'inverted' target base pairs of triplexes in the pyrimidine/parallel motif.

Eight base analogs were tested as third strand residues in otherwise homopyrimidine strands opposite each of the 'direct' (A.T and G.C) and 'inverted' (T.A and C.G) Watson-Crick base pairs, using UV melting profiles to assess triplex stability. The target duplexes contained 20 A.T base pairs and a central test base pair X.Y, while the third strand contained 20 T residues and a central Z test base. Z included 5-bromo-uracil, 5-propynyluracil, 5-propynylcytosine, 5-methyl-cytosine, 5-bromocytosine, hypoxanthine, 2-amino-purine and 2,6-diaminopurine. Some of the base analogs enhanced third strand binding to the target duplex with one or other 'inverted' central base pair relative to the binding afforded by any of the canonical bases. Other analogs did the same for the duplexes with the 'direct' target pairs. The increasing order of triplex stabilization by these base analogs is: opposite the 'inverted' base pairs, for T.A, A < C < 5-pC < 5-pU < T < 5-BrC < 5-meC < 5-BrU < 2-AP < 2,6-DAP < Hy < G, for C.G, 2-AP < A < Hy < G < 5-pC < 5-BrC < 5-meC < C < 2,6-DAP < T < 5-BrU < 5-pU; opposite the 'direct' base pairs, for A.T, 2-AP < A < 5-meC < C < G < Hy < 2,6-DAP < 5-pU < T = 5-BrU < 5-BrC < 5-pC, for G.C, G < 2,6-DAP < 2-AP < A < Hy < T < 5-BrU < 5-pU < 5-pC < 5-BrC < C < 5-meC.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

PREDICTOR: a web-based tool for the prediction of atomic structure from sequence for double helical DNA with up to 150 base pairs

We announce the release of a web-based tool for DNA structure prediction (PREDICTOR) which allows the calculation of atomic structures of double-helical DNA with up to 150 Watson-Crick base pairs (http://farwer.staff.shef.ac.uk/ PREDICTOR). The semi-empirical method uses computational chemistry to extrapolate knowledge of sequence-dependent DNA structure contained in the X-ray crystal structure database. The properties of the base stacking interactions are treated theoretically, and an empirical model is used to add the conformational constraints imposed by the backbone. For DNA oligomers in the X-ray crystal structure database that were not used for parameterisation of the model, the method distinguishes A and B form DNA reasonably reliably, and the final structures are accurate to 2 ? rmsd. Simulation of a 150mer and a 494mer with experimentally confirmed bending clearly reproduces the bending whereas the predicted structure of a random 150mer does not show any curvature. Calculation times are 90 seconds for an octamer and 7 minutes for a 30mer.     

Immobilized carboxypeptidase A as a probe for studying the thermally induced unfolding of bovine pancreatic ribonuclease.

A method for the preparation of Sephadex-immobilized carboxypeptidase A is presented. This form of the enzyme has the same specific activity as the soluble enzyme at room temperature, but retains its activity at higher temperatures (60-70 degrees). This preparation of immobilized carboxypeptidase A was used, as a proteolytic probe, to investigate the thermally induced unfolding of the C-terminus of ribonuclease A. This technique indicates that the C-terminal residues of ribonuclease A do not unfold until the high-temperature region of the thermal transition (as determined by ultraviolet difference spectrophotometry and optical rotation).