An improved method for the detection of genetic variations in DNA with denaturing gradient gel electrophoresis

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA:DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that employment of RNA:DNA duplexes makes DGGE much more practical for screening a large number of samples than that of DNA:DNA heteroduplexes originally developed by Lerman et al. (1986), because preparation of RNA probes is easier than that of DNA probes. Three different 32P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA:DNA duplexes were examined by DGGE. The presence of mismatch(es) was detected as a difference in mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and 3 thalassemic human beta-globin genes. The results of the experiments on the cloned DNAs suggest that DGGE of RNA:DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 of IVS2 (IVS2-666) of the human beta-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was made in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for the detection of heritable variation and should be a powerful tool for the detection of fresh mutations in DNA, which occur outside the known restriction sites.     

RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes

It was reported that RNase HII from Chlamydia pneumoniae (CpRNase HII) had RNase H activity on RNA/DNA duplex. We have analyzed the cleavage specificity of CpRNase HII on DNA-rN1-DNA/DNA duplex (rN1, one ribonucleotide). Various mismatches were introduced into the DNA-rN1-DNA/DNA duplexes at or around the ribonucleotide. The mismatches of duplexes resulted in slower cleavage rates compared to the matched duplexes. Furthermore, a greater reduction in cleavage activity was observed for the mismatches located at or adjacent to the ribonucleotide. The mismatches at the same position of DNA-rN1-DNA/DNA duplexes have different impact on the cleavage rates of CpRNase HII depending on the types of mismatches. These findings may offer further insights into the physical binding and catalytic properties of CpRNase HII-substrate interaction.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition.

alpha and beta-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12: the observed melting temperatures are 27 degrees C for beta-oligodeoxyribonucleotide/RNA hybrid and 53 degrees C for alpha-oligodeoxyribonucleotide/RNA. alpha-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5'-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:alpha- or beta-d(G2T12G2) hybrids or on mRNA:alpha- or beta-oligonucleotides hybrids. Specific hybridization protects RNA strech when using alpha-oligonucleotides but not beta-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in alpha-oligodeoxyribonucleotides:RNA duplexes.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases

Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells. Received: 14 November 1996 / Accepted: 17 February 1997     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Detection of single base mismatches of thymine and cytosine residues by potassium permanganate and hydroxylamine in the presence of tetralkylammonium salts.

In the presence of tetramethylammonium chloride, potassium permanganate specifically modifies mismatched thymines. Similarly, the modification of mismatched cytosines by hydroxylamine was enhanced by tetraethylammonium chloride. Modification followed by piperidine cleavage permits specific identification of the T and C mismatches and by extension, when the opposite DNA strand is analyzed, of A and G mismatches as well. These reactions can be performed conveniently with DNA immobilized on Hybond M-G paper. We describe conditions that exploit these reactions to detect mismatches, e.g. point mutations or genetic polymorphisms, using either synthetic oligonucleotide probes or PCR amplification of specific genomic DNA sequences.     

Hybridase cleavage of RNA. V. Complementary precision of cleavage]

The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.     

Chemically modified oligonucleotides with efficient RNase H response

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.     

The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.     

Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes.

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.     

Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage

Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity. In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix. To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI). Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes. PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions. Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides. In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific. Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III. E. coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage. On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity. PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site. These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix.     

Amplification and characterization of a beta-globin gene synthesized in vitro.

Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.