Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.     

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.     

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.     

A microanalytical procedure for determination of the base composition of DNA

A new procedure for the determination of the percentage guanine plus cytosine (% G+C; mol/100 mol) values of microquantities of DNA is described. Its principle is a DNA-polymerase-I-directed nick translation of DNA in the presence of dGTP, dTTP, [3H]dCTP, and [alpha-32P]dATP. Kinetics experiments indicate that the plateau value is reached in about 20 min of incubation under our experimental conditions. Percentage G+C is obtained from the linear relation 1/(% G+C) = 0.01 K [32P]/[3H] + 0.01, where the ratio of trichloroacetic-acid-precipitable radioactivity is taken into account, the K value being determined for each experiment by using a few reference DNAs of known composition. This procedure has proven suitable for analysis of plasmidic, viral and cellular DNAs of different base composition (25-75% G+C), shape (linear and circular double-stranded DNA) and size (100-150 000 base pairs). Usual methods for % G+C analysis (buoyant density and melting temperature determinations) yield unreliable results in the presence of either modified or unusual bases: the double-labeling procedure is still valid under these conditions. The latter is, therefore, the method of choice for analysis or rare DNA species which are available in very small quantities (it requires amounts of DNA as low as 1 ng, i.e. several order of magnitude lower than those used for chromatographic analysis of DNA hydrolysates). Since the obtention of highly purified DNA is an essential prerequisite for the double-labeling procedure, a method for purification of bacterial DNA is detailed in the present work.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA.

1. The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes. In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition. The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag+. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.     

A mutant of Mycoplasma mycoides subsp. mycoides lacking the H2O2-producing enzyme l-α-glycerophosphate oxidase

A universal rapid procedure to determine the DNA base composition (mol% guanine + cytosine) of Gram-positive bacteria is described. Cells of Gram-positive bacteria were lysed with achromo-peptidase and the mol% G + C of their DNAs were determined by using high performance liquid chromatography. One ml of a Gram-positive bacterial suspension which matched MacFarland No. 3 standard turbidity was sufficient to determine the mol% G + C within 3 h.     

Spin-labelling of DNA with hydrazine mustard spin label (HMSL)

1. The hydrazine mustard spin label (HMSL), recently synthesized in our laboratory (Raikova, 1977) was used for spin-labelling of DNA. 2. It alkylates both double- and single-stranded DNAs. 3. The reaction of HMSL with DNA was studied with respect to the kinetics of alkylation, dependence on salt concentration and base specificity. 4. It was found that HMSL is a base-specific reagent, alkylating preferentially guanine. According to their ability to bind HMSL, the four deoxyribonucleotides are ordered in the following way: G greater than A greater than C greater than T. 5. The EPR spectra obtained strongly depended on the secondary structure of the spin-labelled DNA: unlike the immobilized spectra of the double-stranded DNAs (2AZZ = 44.8G), the EPR spectra of single-stranded DNAs were non-immobilized (2AZZ = 32.8 G). 6. When sheared double-stranded DNA was spin-labelled, the parameters of the EPR spectrum depended also on the GC content of DNA.     

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.     

In vitro selection of DNAs with an increased propensity to form small circles

A protocol was devised to select for DNA molecules that efficiently form circles from a library of 126 base pair DNAs containing 90 randomized base pairs. After six rounds of selection, individual molecules from the library showed 20‐ to 100‐fold greater j‐factors compared with the starting library, validating the selection protocol. High‐throughput sequencing revealed a sinusoidal pattern of enrichment and de‐enrichment of A/T dinucleotides in the random region with a 10.4 base pair period associated with the helicity of DNA. A similar, but more moderate pattern of C/G dinucleotides was offset by precisely half a helical turn. While C/G dinucleotide enrichments were evenly distributed, A/T dinucleotide enrichments displayed a preference to cluster in individual DNA molecules. The most highly enriched 10 base pair sequences in the random region contained adjacent blocks of A/T and C/G trinucleotides present in some, but not all, rapidly cyclizing molecules. The phased dinucleotide enrichments closely match those present in accurately mapped yeast nucleosomes, confirming the importance of DNA bending in nucleosome formation. However, at certain sites the nucleosomal DNAs show dinucleotide enrichments that differ substantially from the cyclization data. These discrepancies can often be correlated with sequence specific contacts that form between histones and DNA. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 303–320, 2015.     

Mechanisms of chromosome banding

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.     

Mitochondrial DNA of Yoshida ascites tumour cells. Physicochemical properties and biological function.

Mitochondrial DNA from Yoshida A.H. 130 cells, has been characterized by determination of the buoyant density by CsCl equilibrium density gradient centrifugation and the thermal denaturation and renaturation behaviour. These studies have been carried out parallelly on nuclear DNA from the same cells in order to search for possible differences between both DNAs. Mitochondrial DNA of Yoshida cells presents an equilbrium in CsCl of 1.7154 g/cm3 and a sharp melting with a Tm of 92 degrees C. Nuclear DNA presents an equilibrium of 1.7030 g/cm3 and a Tm of 88 degrees C. The guanine plus cytosine content in both DNAs has been calculated from tumour results and compared with the content in normal rat liver cells M-DNA of tumour cells presents a higher guanine plus cytosine content than N-DNA, whereas in normal liver cells is higher in N-DNA. N-DNAs of both normal and tumour cells have the same guanine plus cytosine content, whereas M-DNA from tumour cells presents a significant increase (about 35%) with regard to this from normal liver cells.     

Evidence of antisense tumor targeting in mice

Even though increased accumulations of radiolabeled antisense DNAs compared to control DNAs are becoming a routine observation in cultured tumor cells, trustworthy evidence of tumor targeting in vivo by an antisense mechanism remains elusive. The goal of this study was to obtain convincing evidence of antisense tumor targeting in nude mice by using two different tumors and both intratumoral (i.t.) and intravenous (i.v.) administration of radiolabeled antisense and control sense DNAs. Both the MDR++ cell line KB-G2 and its parent MDR+ cell line KB-31 were used in this study. The antisense (AS) DNA was directed against the AUG start codon of the MDR1 mRNA and, along with the sense (S) control DNA, was a uniform phosphorothioate administered naked. In previous cell culture studies from our laboratories, the accumulation of this AS DNA was strikingly high in KB-G2 cells and only average in KB-31 cells, a fact we attribute to the 1000-fold higher expression by RT-PCR of MDR1 mRNA in the former cell line. In this study, both DNAs were radiolabeled with (99m)Tc via MAG3 and administered i.t. or i.v. at 1 microg (100 microCi) per animal 24 h prior to sacrifice and dissection in mice bearing thigh tumors of about 1 g. Following i.t. administration, no statistically significant differences (Student's t test, p < 0.05, N = 4) between the AS and S DNA biodistributions in normal tissues were observed except in the KB-G2 mice in which muscle levels were lower for the S control. In contrast, tumor levels in the KB-G2 animals were significantly higher for the AS DNA vs S DNA (14.7 vs 8.5% ID/g) while this difference (8.6 vs 4.3% ID/g) was insignificant in the KB-31 animals. The whole body images obtained just prior to sacrifice clearly show improved targeting of AS DNA vs S DNA in the KB-G2 but not the KB-31 animals. Calculations based on these results show that about 60 000 AS DNAs accumulated specifically (i.e. AS DNA - S DNA) per KB-G2 tumor cell following i.t. administration. When administered i.v. rather than i.t., higher tumor levels in KB-G2 animals compared to KB-31 were not observed, most likely because of the lower dosage reaching the tumors. When the KB-G2 and KB-31 results are combined, no statistically significant differences between the AS and S DNA biodistributions in normal tissues were observed except in blood in which S DNA levels were higher and in spleen in which they were lower. In contrast, tumor levels were significantly higher for the AS DNA vs S DNA (0.100 vs 0.063% ID/g). Calculations based on these results show that about 400 AS DNAs accumulated specifically per tumor cell following i.v. administration. Therefore evidence for tumor targeting in vivo by an antisense mechanism has been obtained in that statistically higher tumor accumulations of the (99m)Tc-AS DNA were observed compared to the control (99m)Tc-S DNA both following i.t. and i.v. administrations. The successful localization of AS DNA in tumor demonstrates that in vivo AS targeting of tumor is feasible although improvements in tumor delivery and normal tissue clearance are needed for practical antisense imaging.     

The heterogeneity of B-chromosome DNA: no evidence for a B-chromosome specific repetitive DNA correlated with B-chromosome effects on meiotic pairing in the triticinae

The compositional heterogeneity of DNAs of A (normal) and B (supernumerary) chromosomes of Aegilops speltoides, Ae. mutica and Triticum aestivum has been compared in order to elucidate the mechanism of B-chromosome disruption of meiotic pairing in interspecific hybrids. Comparisons of % heterologous association after DNA/DNA hybridation at C₀t 10^?2 (highly repetitious DNA) and C₀t 100 (moderately repetitious DNA), and comparisons of nucleotide base divergence (ΔT_ms) and thermal elution profiles of homologous and heterologous duplexes, show that genotypes of Aegilops spp., having large numbers of Bs, do not carry additional families of repetitious DNA exclusive to B-chromosomes. Neither the presence of Bs nor the direction of DNA/DNA hybridisation affect the above parameters. No cryptic DNA satellites were revealed in A- and B-chromosome DNA after sedimentation in actinomycin D-CsCl gradients; and there were no significant differences in buoyant densities of main-band DNA. Mean melting temperatures (T_m); transition temperatures (ΔT) and numbers and positions of peaks of dissociating DNA fractions in profiles of differentiated melting curves of native DNAs were similar in strictly comparable denaturation conditions. One small AT-rich (< 5%) DNA fraction correlated with speltoides Bs was revealed; however, no corresponding fraction is associated with mutica Bs. The overall similarity in numbers and base composition of families of DNA (repetitious and unique) of As and Bs is discussed in relation to the origin of Bs and the origin of the meiotic diploidising system in haploid T. aestivum.     

Cryptic satellites rich in inverted repeats comprise 30% of the genome of a hermit crab

One major very highly repeated (VHR) DNA (approximately 7 X 10(6) copies/genome; repeat unit = 156 base pairs (bp)), a family of three minor VHR DNAs (approximately 2.8 X 10(6) copies/genome; repeat units = 71-74 bp), and a number of trace components account for almost 30% of the genome of a hermit crab. The repeat units of the three minor variants are defined by identical 14-bp G + C-rich inverted repeats that might form cruciforms. Two copies of the repeat unit (CCTA) of one of two patent satellites of this crab (Skinner, D. M., and Beattie, W. G. (1974) Biochemistry 13, 3922-3929; Skinner, D. M., Beattie, W. G., Blattner, F. R., Stark, B. P., and Dahlberg, J. E. (1974) Biochemistry 13, 3930-3937) occur at the center of one in seven of the G + C-rich inverted repeats; copies of the other patent satellite (Chambers, C. A., Schell, M. P., and Skinner, D. M. (1978) Cell 13, 97-110) are found in main component DNA. The sequences of both the major and minor VHR DNAs are characterized by short tracts of An and/or Tn (n = 4-7) residues whose presence would permit the formation of perfectly matched stems separated by loops of 8-16 bp. The An and/or Tn tracts are interspersed with segments of G + C-rich DNA and are arranged differently in the major and minor VHR DNAs. Although the repeat units of the major and the three minor VHR DNAs are arranged in tandem, the composition and sequence of their bases are such that they do not form distinct bands in CsCl gradients; they are cryptic satellites.     

Effect of DNA base composition on the intercalation of proflavine. A kinetic study.

The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.