Biomedical subjects
Publications and source records attributed to J H van Boom.
Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].
NMR studies were carried out on some alternating pyrimidine-purine sequences: the single-stranded tetramers CACA and UGUG and the self-complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann-Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single-stranded stack in equilibrium with random coil and the duplex in equilibrium with random coil equilibria. The populations of N-type conformer of the ribose rings were estimated from the observed J1'2'. Comparisons with another alternating pyrimidine-purine sequence Um2(6)AUm2(6)A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given. Previous 1H-NMR investigations of Um2(6)AUm2(6)A revealed that the population of bulge-out structure diminishes compared to m2(6)AUm2(6)A due to the U(1)-m2(6)A(2) stacking interaction. In CACA a strong stacking proclivity (Tm = 310 K) together with a clear preference for N-type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (Tm = 288 K) and a bias towards S-type sugar is present. Besides a small amount of stack, a significant contribution of bulge out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge-out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the Tm values of CACA and d(CACA), whereas the Tm of UGUG (288 K) is much lower than the Tm of d(TGTG) (315 K). It is suggested that the absence of bulge-out structures in DNA purine-pyrimidine-purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues. The Tm values (average 341 K) for the duplex in equilibrium with random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.
NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.
It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.
The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)
NMR and CD studies were carried out on the dinucleotides 5'-methylphospho-N6-dimethyladenylyl-uridine (mpm62-U) and 5'-methylphospho-uridylyl-N6-dimethyladenosine (mpU-m62A) and on the trinucleotide U-m62A-U. A detailed comparison is given of the conformational features of mpm62A-U and mpU-m62A with the corresponding 5'-nonphosphorylated dinucleotides m62A-U and U-m62A, respectively. The behaviour of the trinucleotide U-m62A-U is compared with the properties of the constituent dinucleotides U-m62A and mpm62A-U. Chemical-shift and CD data were used to determine the amount of stacking interactions. For each compound NMR spectra were recorded at two or three sample concentrations in order to separate intermolecular and intramolecular base-base interactions. The coupling constants of the ribose ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta, gamma and epsilon are presented. The combined data indicate a strong similarity between mpm62A-U and m62A-U both in degree and in mode of stacking. In contrast, the existence of different types of stacking interactions in mpU-m62A and U-m62A is suggested in order to explain the NMR and CD data. It is concluded that dinucleoside bisphosphates serve better as a model for the behaviour of trinucleotides than dinucleoside monophosphates. The trinucleotide U-m62A-U adopts a regular single-stranded stacked RNA structure with preference for N-type ribose and gamma+ and beta t backbone torsion angles. The difference in behaviour between the U-m62A- part of U-m62A-U and the dimer U-m62A is seen as a typical example of conformational transmission.
A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.
Two sets of products are formed from DNA upon treatment with Fe(II).bleomycin + O2. One set, which is believed to derive from a C-4' hydroperoxy derivative of the DNA deoxyribose moiety, includes the four possible base propenals, as well as DNA oligomers having deoxynucleoside 3'-(phosphoro-2"-O-glycolates) at their 3'-termini. The other set of products consists of free bases and alkali-labile lesions, the latter of which had not previously been characterized structurally. By use of the self-complementary dodecanucleotide d(CGCTTTAAAGCG) having a site modified by Fe-bleomycin three nucleotides from the 5'-end, it has been possible to characterize the alkali-labile product as a C-4' hydroxyapurinic acid. When the bleomycin-treated dodecanucleotide was treated with agents that effected decomposition of the alkali-labile lesion, products of the form CpGpx were obtained, and these proved useful for structural characterization of the alkali-labile lesion. Treatment with alkali produced CpGpx, where x was 2,4-dihydroxycyclopentenone. Alternatively, treatment with hydrazine provided a pyridazine derivative, and aqueous alkylamines led to formation of CpGp itself. The structures of all dinucleotides produced from the alkali-labile lesion were verified by direct comparison with authentic synthetic samples.
Aspergillus and Penicillium species produce extracellular polysaccharides which are immunologically active. Methyl beta-D-galactofuranoside interferes with the reaction between the polysaccharide antigens and the antibodies raised in rabbits. Of the different interlinked dimers of beta-D-galactofuranosides (1----2; 1----3; 1----5; 1----6) the (1----5) interlinked beta-D-galactofuranoside gave the highest inhibition. An increasing inhibitory effect of di-, tri-, tetra-, penta-, hexa-, and heptamer of (1----5) interlinked beta-D-galactofuranosides was observed. It was noticed that the penta-, hexa- and heptamer of (1----5) interlinked beta-D-galactofuranosides were able to link antibodies raised against the extracellular polysaccharides produced by Penicillium species. The tetramer molecule was able to neutralize the binding of antibodies, which are naturally present in human sera, to the polysaccharides produced by Penicillium and Aspergillus species.
Irradiation of the double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with UV light causes dimerization of the two central thymine residues. Proton NMR data reveal that this photodimer has the same chemical structure as the photodimer, which is formed upon UV irradiation of the single strand d(GCGTTGCG), a cis-syn-cyclobutane-type thymine dimer. Irradiation of the purified thymine dimer double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with visible light in the presence of photoreactivating enzyme isolated from Anacystis nidulans leads to an increase in absorbance at 260 nm, which is characteristic for the repair of thymine dimers. The NMR spectrum recorded after the photoreactivating treatment indeed shows that a complete conversion to the starting octamer has occurred.
We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 +/- 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.
Recycling of a number of glycoproteins along the site of action of mannosidase I (the distal endoplasmic reticulum/cis-Golgi) was followed in several different cell lines. Treatment of cells with 1-deoxymannojirimycin (dMM) produced high mannose oligosaccharides at positions otherwise occupied by complex-type oligosaccharides in these glycoproteins. Conversion of high-mannose-type oligosaccharides to complex oligosaccharides of proteins initially synthesized in the presence of dMM was used as a marker for recycling of glycoproteins along the site of action of dMM. In contrast to findings reported by Snider and Rogers (Snider, M. D., and O. C. Rogers. 1986. J. Cell Biol. 103:265-275), removal of dMM did not result in reconversion of high-mannose oligosaccharides to complex-type sugars, even after prolonged periods of culture. We conclude that surface glycoproteins do not recycle through the cis-medial Golgi elements.
Analysis of nuclear Overhauser enhancements in two-dimensional NMR spectra of the complex of lac repressor headpiece with a 14 base pair lac operator fragment shows that the second helix of the headpiece binds in the major groove of DNA, as has been suggested. The orientation of this helix is approximately 180 degrees different from the proposed models and from that found in the X-ray structure of the 434 repressor-operator complex. The model of the lac headpiece-operator complex provides a good explanation for a large amount of biochemical, genetic and physical data.
By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.
The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non-enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon-anticodon interaction.
The individual diastereomers of trimer A2'p5'A2'p(s)5'A, containing one phosphorothioate linkage, were prepared via a modified hydroxybenzotriazole phosphotriester approach. The 5'-phosphorylated derivatives of the latter compounds were obtained after phosphorylation with a 6-trifluoromethyl-1-benzotriazolyl activated phosphoromorpholidate.
NMR studies were carried out on samples of the non-self-complementary tetramers d(C-A-C-A), d(T-G-T-G), d(G-A-G-A) and d(T-C-T-C) and of 1:1 mixtures of the complementary tetramers d(C-A-C-A) X d(T-G-T-G) and d(G-A-G-A) X d(T-C-T-C) at two DNA concentrations and of the self-complementary octamers d(C-A-C-A-T-G-T-G) and d(G-A-G-A-T-C-T-C). Assignments, based upon one-dimensional NOE and homonuclear-decoupling and two-dimensional correlated and NOE spectroscopies are given of the resonances of most of the base and sugar protons. Chemical shift vs temperature profiles, constructed for all samples, yielded insight into the temperature- and concentration-dependent conformational behaviour of the compounds and were used to obtain thermodynamic parameters pertaining to the stacked-single-strand----random-coil and duplex----random-coil equilibria. Vicinal proton-proton couplings were analyzed in terms of the conformation of the deoxyribose rings in the single-stranded tetramers and duplexed octamers. The NOE patterns, chemical shift profiles, imino-proton resonances and coupling data revealed that the compounds adopt B-DNA-like structures. The ratio duplexed/stacked-single-strand/random coil depends upon external conditions as well as upon base sequence. The thermodynamic data indicate that: in terms of single-helical stacking, the R-R steps (Tm 321-328 K) appear more stable than the Y-R or R-Y steps (Tm 308-316 K) and the Y-Y steps score least (Tm 290-300 K), and the duplexes consisting of alternating, d(Y-R)n, strands are more stable, in terms of delta H degrees, compared to the d(R-R)n X d(Y-Y)n duplexes. The analyses of the couplings demonstrated that the sugars of the single-stranded tetramers and duplexed octamers occur as a blend of N- and S-type conformers, with a preference for the S-type (C2'-endo) sugar conformation: upon duplex formation, no significant shift in the N-type/S-type ratio was observed. The fraction S-type sugar conformation of a given residue, %S, in the stacked-single strands was found to depend upon the nature of its own base and that of the adjacent residues: sugars in an R-R stretch display high values of %S (90-100), whereas those in Y-Y stretches show relatively low values (approximately equal to 65).(ABSTRACT TRUNCATED AT 400 WORDS)
Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.