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PubMed1982

Studies on deoxyribonucleic acids and related compounds. V. Synthesis of pentadecanucleotide duplex containing the ideal Pribnow sequence of promoter by the phosphotriester method using a new phosphorylating reagent.

A phosphorylating reagent o-chlorophenyl phosphoro-p-anisi-dochloridate was synthesized to phosphorylate the 3'-hydroxyl group of N, 5'-protected deoxynucleosides. These nucleotides served as 3'-terminal units for the synthesis of oligonucleotide blocks. By condensation of these oligonucleotide blocks the partially complementary deoxypentadecanucleotides dAGCTTATAATGC-TCG and dAGCTCGAGCATTATA, which contained the ideal Pribnow sequence TATAATG, were synthesized.

Base Sequence↗
PubMed1976

Restriction endonucleases.

This review provides a comprehensive account of the current status of the biology and biochemistry of restriction endonucleases. Both Class I and Class II restriction endonucleases will be considered. However, emphasis will be placed on the Class II group, which recognizes and cleaves a specific duplex DNA sequence. Their occurrence, purification, and characterization is discussed in detail. The characterization includes physical mapping information and determination of recognition sequences. In addition to detailed discussions of the biochemical properties of the enzymes, considerable attention is paid to the uses of these enzymes as tools for research in molecular biology. These uses include physical mapping of genomes and their transcripts, genetic analysis (marker rescue, etc.), DNA sequence analysis, analysis of complex genomes, and genetic engineering. Specific examples of each use are outlined. Practical aspects of both the isolation and use of the restriction endonucleases form the major theme of this review.

Bacteria↗
PubMed1984

Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum.

The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes. The proton resonances in the 1H NMR spectra of the oligo[r(C-G)] duplexes were assigned by nuclear Overhauser effect experiments. The chemical shift-temperature profiles of the base proton signals and the sharp singlets observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. Moreover, a 500-MHz two-dimensional nuclear Overhauser effect experiment recorded for r(C-G-C-G-C-G) shows that all guanine bases adopt the normal anti-conformation. CD-temperature profiles and 31P NMR spectra of oligo[r(C-G)]s support this conclusion. These results indicate that duplexes of oligo- and polyribonucleotides containing alternating C-G sequences can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.

Base Sequence↗
PubMed1982

Evolution of human Y-chromosome DNA.

We have used human male-specific 3.4 kb Hae III restriction endonuclease fragments to explore the evolutionary history of man's Y-chromosome. We have identified four sets of reiterated sequences on the basis of their relative sequence homology with autosomal DNA. The sequences account for approximately 40% of the human Y-chromosome, are interspersed within the same 3.4 kb Hae III fragments, are heterogeneous and contain all reiterated DNA previously demonstrated to be specific for the Y-chromosome (it-Y DNA). Y-specific 3.4 kb Hae III sequences do not reassociate with either human female or ape DNA at standard reassociation criteria. However, approximately half of it-Y DNA (cross reacting it-Y) reassociates with both human female and ape DNA at reduced reassociation criteria. The remaining half (Y-specific it-Y) retains its specificity for the human Y-chromosome. These two sets of it-Y DNA have distinct reiteration frequencies and thermal stabilities with their Y-chromosome homologs. Non-Y-specific 3.4 kb Hae III sequences reassociate with both human female and ape DNA at standard reassociation criteria. The abundance of these non-Y-specific sequences decreases as a function of their evolutionary distance for man. One subset of non-Y-specific 3.4 kb Hae III sequences forms stable duplexes with human Y-chromosome DNA and with human and ape autosomal DNA. No detectable base-mismatch occurs among these homologs suggesting complete conservation of these sequences during primate evolution. The second subset of Non-Y-specific Hae III sequences form stable duplexes with human Y-chromosome DNA but highly mismatched duplexes with human and ape autosomal DNA. The finding that homologs of 3.4 kb Hae III sequences are not found within the Y-chromosome of apes but are only present in autosomes suggests that 3.4 kb Hae III sequences are largely autosomal in origin. Since autosomal homologs of most 3.4 kb Hae III-sequences exhibit a greater degree of divergence that those localized to the Y-chromosome, their evolutionary history seems to be chromosome-dependent. Our findings are not easily correlated with the comparative morphology of primate Y-chromosomes and suggest that sequence rearrangement has been a major event in the evolution of the human Y-chromosome. The significance of the specific interspersion of four sets of reiterated sequences, with distinct evolutionary histories, within a repeating unit specific to the human Y-chromosome is not clear. The apparent conservation of at least some of these reiterated sequences suggests they may be of functional importance.

Animals↗
PubMed1989

Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5' end of an 11-mer homopyrimidine oligonucleotide of sequence d(TTTCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used as a substrate for the phenanthroline-oligonucleotide conjugate. In the presence of copper ions and a reducing agent, a single specific double-strand cleavage site was observed at 20 degrees C by agarose gel electrophoresis. The efficiency of double-strand cleavage was greater than 70% at 20 degrees C and pH 7.4. Secondary cleavage sites were observed when binding of the oligonucleotide to mismatched sequences was allowed to take place at low temperature. The exact location of the cleavage sites was determined by polyacrylamide gel electrophoresis of denatured fragments by using both simian virus 40 DNA and a synthetic DNA fragment containing the target sequence. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove even though the phenanthroline linker was located in the major groove. Linkers of different lengths were used to tether phenanthroline to the oligonucleotide and their relative efficacies of DNA cleavage were compared. Based on these comparative studies and on model building, it is proposed that the phenanthroline ring carried by the oligonucleotide intercalates from the major groove and that copper chelation locks the complex in place from within the minor groove where the cleavage reaction occurs.

Base Sequence↗
PubMed1990

N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts.

Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.

Base Sequence↗
PubMed1988

Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome.

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.

Adenine Nucleotides↗
PubMed1992

Crystal and molecular structure of d(GTGCGCAC): investigation of the effects of base sequence on the conformation of octamer duplexes.

The structure of the self-complementary deoxyoctanucleotide d(GTGCGCAC), which crystallized as an A-type helix in the space group P4(3)2(1)2, with one strand in the crystallographic asymmetric unit has been determined and refined to a final R-value of 0.154 using 1.64-A diffraction data collected on an area detector. In contrast to the closely related sequence d(GTGTACAC)tet, there was no evidence for an ordered spermine molecule in the major groove of this octamer. Ordered water is found associated with almost all the exposed hydrogen bonding groups of the octamer. A pentagonal ring of water molecules is hydrogen bonded to O6 and N7 of G3 and the N4 and O6 of the C4.G13 base pair. A detailed comparison of the local helical parameters of d(GTGCGCAC) and d(GTGTACAC)tet is presented. The base sequence change at the center of the octamers affects several of the local helical parameters, via both intra- and interduplex interactions within the crystal.

Base Composition↗
PubMed1990

NMR studies of the interaction of chromomycin A3 with small DNA duplexes. Binding to GC-containing sequences.

The interaction of chromomycin A3 with the oligodeoxyribonucleotides 1, d(ATGCAT), 2, d(ATCGAT), 3, d(TATGCATA), and 4, d(ATAGCTAT), has been investigated by 1H and 31P NMR. In the presence of Mg2+, chromomycin binds strongly to the three GC-containing oligomers 1, 3, and 4 but not to the CG-containing oligomer 2. The proton chemical shift changes for 1 and 3 are similar, and these DNA duplexes appear to bind with a stoichiometry of 2 drugs:1 Mg2+:1 duplex. The same stoichiometry of 2 drugs:1 duplex is confirmed with 4; however, proton chemical shift changes differ. An overall C2 symmetry is exhibited by the drug complex with 1, 3, and 4. At a molar ratio of 2.0 (drugs:duplex), no free DNA proton NMR signals remain. Two-dimensional nuclear Overhauser exchange spectroscopy (NOESY) of the saturated chromomycin complex with 1 and 3 positions both chromomycinone hydroxyls and the E carbohydrates in the minor groove and provides evidence suggesting that the B carbohydrates lie on the major-groove side. This is supported by several dipolar coupling cross-peaks between the drug and the DNA duplex. Drug-induced conformational changes in duplex 1 are evaluated over a range of NOESY mixing times and found to possess some characteristics of both B-DNA and A-DNA, where the minor groove is wider and shallower. A widening of the minor groove is essential for the DNA duplex to accommodate two drug molecules. This current minor-groove model is a substantial revision of our earlier major-groove model [Keniry, M.A., Brown, S.C., Berman, E., & Shafer, R.H. (1987) Biochemistry 26, 1058-1067] and is in agreement with the model recently proposed by Gao and Patel [Gao, X., & Patel, D. J. (1989a) Biochemistry 28, 751-762].

Base Composition↗
PubMed1987

Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence.

Circularization and reverse circularization of the Tetrahymena thermophila rRNA intervening sequence resemble the first and second steps in splicing, respectively. However, site-specific base substitutions show that different nucleotides are involved in selection of the 5' splice site and the circularization sites. Furthermore, a substitution at the major circularization site that prevents circularization can be suppressed by second substitutions at two different nucleotide positions. A model is proposed in which adjacent and overlapping sequences can function as a binding site, forming a short duplex with the sequence at the circularization site and thus directing circularization and reverse circularization. Because the 5' exon-binding site and three potential circularization binding sites fall within a contiguous eight nucleotide region, this sequence may translocate relative to the catalytic core of the ribozyme in a template-like manner.

Animals↗
PubMed1991

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1988

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed.

Animals↗
PubMed1988

Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation.

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (beta) anomer of thymidine or the synthetic (alpha) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA.dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both alpha and beta oligothymidylates can recognize a DNA double helix containing an oligo(dA).oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) alpha oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, beta oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine.oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.

Cross-Linking Reagents↗
PubMed1991

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1989

Spectroscopic properties and helical stabilities of 25-nt parallel-stranded linear DNA duplexes.

DNA strands with appropriate sequences of dA and dT can form a stable duplex in which the two strands adopt a parallel (ps) instead of the conventional antiparallel (aps) orientation. Four 25-nt dA.dT-containing deoxyoligonucleotides (D1-4) were synthesized. D1 has the sequence 5'-dA10TA2T4A3TAT3-3'. Viewed with the same polarity, D2, D3, and D4 are the complement, inverted complement, and inverse of D1, respectively. The two combinations D1.D3 and D2.D4 form conventional antiparallel duplexes (aps-D1.D3, aps-D2.D4). D1.D2 and D3.D4, however, constitute stable parallel-stranded duplexes (ps-D1.D2, ps-D3.D4), as established by various criteria including the following: (i) The electrophoretic mobilities of ps-D1.D2 and ps-D3.D4 are similar to those of the antiparallel-stranded duplexes. (ii) The ultraviolet absorption and circular dichroism spectra of the ps duplexes are indicative of a base-paired structure, but differ systematically from those of the aps helices. (iii) Similar salt-dependent thermal transitions are observed for the four duplexes, but the melting temperatures of the ps molecules are lower by 13-18 degrees C.

Base Sequence↗