Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([²H₃]-Methyl)cytosine and [methyl-²H₃]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.     

Incorporation of phosphorothioate groups into fd and phi X174 DNA.

We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.     

Identification of palindromic sequences recognized by restriction endonucleases, as based on the tabularized sequencing data for seven viral and plasmid DNAs

Computer search of DNA sequences for phages φX174, G4, M13 and fd, plasmids pBR322 and pA03, and virus SV40, was employed to prepare tables specifying the size classes and frequencies of DNA segments located between all possible tetra-, penta- and hexanucleotide palindromes. As described earlier (Fuchs et al., 1978), these tables permit identifying sequences recognized by most of the restriction endonucleases. The effect of sequencing errors on the accuracy of the present identification method is evaluated. Only four of the 224 listed sequences do not appear in any of the seven DNAs, leading to discussion (see Appendix) on the natural sequence distribution.     

Methylated bases in mycoplasmal DNA.

The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed.     

Isolation of specific ribosome binding sites from single-stranded DNA.

The ability of Escherichia coli ribosomes to protect small specific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage f1 and φX174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f1 and φX174 DNA which are protected differ in sequence from each other.When E. coli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of φX174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAase, and a number of additional polypyrimidines.Double-stranded DNAs such as φX174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specific regions as those protected in single-stranded φX174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.     

Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A high-performance liquid chromatographic method to separate five major bases (cytosine, thymine, guanine, adenine, and uracil) and three minor methylated bases (5-methylcytosine, N6-methyladenine, and 7-methylguanine) has been developed using a volatile mobile phase under isocratic conditions. It is extended to quantitate 5-methylcytosine in trace amounts (1 in 20,000 cytosine residues). The suitability of the method has been verified by estimating 5-methylcytosine in DNAs of phi X174 and pBR322. The method has been applied to quantitate the extent of cytosine methylation in DNA of larval silk glands of Bombyx mori. Our results confirm that the pupal DNA of Drosophila melanogaster does not contain detectable amounts of 5-methylcytosine.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Determination of genomic 5-hydroxymethyl-2’-deoxycytidine in human DNA by capillary electrophoresis with laser induced fluorescence

Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine.

Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix.

By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.     

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.     

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.     

Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps.

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions

In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3. We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single stranded DNA, suggesting that bases are stacked in all of the phages. We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages. From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA. The maximum angles were 63° and 64° for the DNAs of class I phages fd and IKe, respectively. The angles were significantly less, 51° and 49°, for the DNAs of the class II phages Pf1 and Pf3, respectively. Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

Correlation between thermal stability maps and genetic maps of double-stranded DNAs

The positional correlation of boundaries of cooperatively melting regions with boundaries of genes and with cleavage sites of restriction enzymes are statistically examined for phiX174, G4, fd, SV40, BKV, and polyoma DNAs. A statistically significant correlation does exist in the case of gene boundaries, but none is detected for cleavage sites.     

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.     

A new method for detection of small modifications in genomic DNA, applied to the human delta-beta globin gene cluster

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].     

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0°C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O-glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82, 289–309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.