Neighboring DNA Research Articles

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations.

During retrovirus replication, a cellularly derived tRNA is annealed to the viral RNA at the primer binding site (PBS) to prime reverse transcription, and both the tRNA and the PBS become copied and matched together on complementary proviral DNA strands prior to integration. Using a viral PBS single base pair mutant which affects provirus expression in undifferentiated cells, we show that reversion to wild type (wt) occurs at a frequency of approximately 50%. Daughter cell lines containing wt or mutant proviruses at identical chromosomal sites have been isolated, supporting a model where an integrated PBS-mismatched provirus was copied before mismatch correction could occur. Virus expression in daughter cells containing the mutant provirus was 100-fold higher than in cells bearing the wt counterpart. Additionally, proviral 5' DNA and cellular 5' flanking DNA became methylated in daughter cells containing wt but not mutant integrants. These results strongly support the current model of retrovirus reverse transcription, and indicate that the wt PBS region contains an element which suppresses virus expression and directs the methylation of viral and neighboring cellular DNA.

Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy.

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.

A source of small repeats in genomic DNA.

The processes of spontaneous mutation are known to be influenced by neighboring DNA. Imperfect nearby repeats in the neighboring DNA have been observed to mutate to form perfect repeats. The repeats may be either direct or inverted. Such a mutational process should create perfect direct and inverted repeats in intergenic DNA. A larger than expected number of direct repeats has generally been observed in a wide range of species in both coding and noncoding DNA. Simulations are carried out to determine how this process might influence the repetitive structure of genomic DNA. These simulations show that small repeats created by this kind of a mutational process can explain the excess number of repeats in intergenic DNA. The simulations suggest that this mechanism may be a common cause of mutations, including single-base changes. The influences of the distance between imperfect repeats and of their degree of similarity are investigated.

Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin.

Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo.

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4s muations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL− hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

A major insertion accounts for a significant proportion of mutations underlying human lipoprotein lipase deficiency.

Lipoprotein lipase (LPL; triacylglyceroprotein acylhydrolase, EC 3.1.1.34) is an important enzyme involved in triacylglycerol metabolism. Primary LPL deficiency is a genetic disorder that is usually manifested by a severe elevation in triacylglycerol levels. We have used a recently isolated LPL cDNA clone to study 15 probands from 11 families with this inherited disorder. Surprisingly, 7 of the probands from 4 families, of different ancestries, had a similar insertion in their LPL gene. In contrast to other human genetic disorders, where insertions are rare causes of mutation, this insertion accounts for a significant proportion of the alleles causing LPL deficiency. Detailed restriction mapping of the insertion revealed that it was unlikely to be a duplication of neighboring DNA and that it was not similar to the consensus sequence of human L1 repetitive elements. This suggests that there must be other mechanisms of insertional mutagenesis in human genetic disease besides transposition of mobile L1 repetitive elements.

A Human Endonuclease Incises Ultraviolet‐Irradiated DNA at Cytosines and Oxidized DNA at Thymines

Both ultraviolet irradiation and oxidation of DNA produce a variety of pyrimidine base damages. A human endonuclease recognizes such altered bases on these DNA substrates. This human endonuclease incises ultraviolet-irradiated DNA exclusively at sites of photochemically modified cytosines. The precise sites of incision by the human enzyme were determined by DNA sequencing. Chemically oxidized DNA was incised exclusively at thymine loci. The degree of enzymic cleavage at cytosine photoproducts was identical at each site. However, the extent of incision at selected oxidized thymine residues varied within the DNA sequence. These results indicate that the distribution of thymine oxidative modifications is influenced by the neighboring DNA bases.

A Brillouin scattering study of the hydration of Li- and Na-DNA films.

AbstractWe have used Brillouin spectroscopy to study the velocities and attenuation of acoustic phonons in wet‐spun films of Na‐DNA and Li‐DNA as a function of the degree of hydration at room temperature. Our data for the longitudinal acoustic (LA) phonon velocity vs water content display several interesting features and reveal effects that we can model at the atomic level as interhelical bond softening and relaxation of the hydration shell. The model for interhelical softening makes use of other physical parameters of these films, which we have determined by gravimetric, x‐ray, and optical microscopy studies. We extract intrinsic elastic constants for hydrated Na‐DNA molecules of c11 ≃ 8.0 × 1010 dynes/cm2 and c33 ≃ 5.7 × 1010 dynes/cm2, which corresponds to a Young's modulus, E ≃ 1.1 × 1010 dynes/cm2 (with Poisson's ratio, σ = 0.44). The negative velocity anisotropy of the LA phonons indicates that neighboring DNA molecules are held together by strong interhelical bonds in the solid state. The LA phonon attenuation data can be understood by the relaxational model in which the acoustic phonon is coupled to a relaxation mode of the water molecules. Na‐DNA undergoes the A to B phase transition at a relative humidity (rh) of 92% while Li‐DNA (which remains in the B form in this range) decrystallizes at an rh of 84%. We find that our Brillouin results for Na‐ and Li‐DNA are remarkably similar, indicating that the A to B phase transition does not play an important role in determining the acoustic properties of these two types of DNA.

Specificity and efficiency of editing of mismatches involved in the formation of base-substitution mutations by the 3'----5' exonuclease activity of phage T4 DNA polymerase.

The specificity and efficiency of base-mispair editing by the 3'----5' exonuclease activity of phage T4 DNA polymerase has been measured using a sensitive infectivity assay. A series of oligodeoxynucleotide primer chains was synthesized chemically. These primers, when hybridized to phi X174 single-stranded DNAs containing an amber codon, result in a mispaired nucleotide at the 3'-hydroxyl end of the primer chain within the amber codon. DNA synthesis on these primer X templates without the removal of the mispaired terminal nucleotide results in the formation of heteroduplex molecules that yield viable revertants upon transfection into an amber nonsuppressor host. This method permits determination of the efficiency of editing of a mismatch to 1 in 10(6) mismatches that escape editing and allows all eight mispairs that can yield viable revertants at an amber codon to be studied. The results of experiments with primers hybridizing to phi X174 am16 and am3 codons show that the order of mispair editing by T4 DNA polymerase is Ttemplate X Gprimer less than (A X G, T X C) less than (T X T, G X A, G X G, A X C) less than A X A. The efficiency of editing depends upon the mispair, as well as the neighboring DNA sequence. Under these conditions of synthesis, the 3'----5' exonuclease activity, depending upon the mispair and DNA sequences beyond the nearest neighbors, is estimated to contribute a factor of from 2.3 X 10(3)- to greater than 10(6)-fold to the accuracy of T4 DNA polymerase.

Genetic organization of plasmid R1162 DNA involved in conjugative mobilization.

DNA involved in the mobilization of broad-host-range plasmid R1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the R1162 map. By examining the transfer properties of plasmids containing cloned fragments of DNA from within this region, we showed that at least four trans-active products and a cis-active site (oriT) were involved in mobilization. A cloned DNA fragment of 155 base pairs was capable of providing full oriT activity. This fragment was located within 600 base pairs of DNA containing the origin of replication of R1162, and its nucleotide sequence and that of neighboring DNA were determined. Activation of oriT required R1162-encoded, trans-acting products. Deletions which resulted in the loss of one or more of these had a variable effect on transfer efficiency and indicated the presence of both essential and nonessential Mob products. Regions encoding these products flanked oriT and in one case appeared to overlap a gene essential for plasmid replication. The implications of these findings with respect to the broad host range of R1162 are discussed.

The sequence and structure of a new serum amyloid A gene.

The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure.

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences.

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.

Preliminary chemical and X-ray studies on the interactions of E. coli DNA with putrescine.

The interactions of putrescine, the major diamine in E. coli, with E. coli DNA as a model of phage DNA were studied by melting temperature analysis, equilibrium dialysis and X-ray diffraction with the aid of molecular model building. The chemical analysis of the DNA-putrescine complex shows that the molar binding ratio of putrescine to DNA (phosphate) is nearly 1 to 2. The equilibrium (or reversible) binding of putrescine to DNA was suggested by the fact that the melting temperature increased according to the concentration of added putrescine, and its elevation was not saturated even at the molar ratio of 6 to 1. The equilibrium dialysis experiments indicate that the association constant for the complex is a little smaller than, but in the same order (10(3) liter/mol) as, that of DNA-spermine complex. The binding of putrescine stabilizes the B-form of DNA fiber, which is well preserved even at 66% relative humidity. The distance between the neighboring DNA helices in the wet fiber increased with the increasing degree of hydration, as in the case of native DNA. Unlike spermine, putrescine does not form precipitate upon mixing with DNA in the concentration range for UV measurement, suggesting that the cross-bridge formed by putrescine is intra-double helical. The equilibrium binding of putrescine to DNA, seems to be important for the life cycle of lambda-phage.

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. III. High resolution thermal denaturation and circular dichroism studies.

High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments. The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC). A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs. This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments. The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R. D., and Wells, R. D. (1982) J. Biol. Chem. 257, 12962-12969). Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C. CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter. The presence of homopolymers had a marked effect on the CD spectra of the fragments.

Effects of Neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. II. In vitro transcription analyses.

Transcription studies were conducted on the primary promoter (which initiates at +1) of lac wild type (wt) and UV5 that had insertions of poly(dA).poly(dT) or poly(dG).poly(dC) in their -60 region. This series of recombinant DNAs was designed to study systematically the effect of neighboring DNA sequences on the lac promoter. Investigations using linear templates showed that wild type expression was enhanced by the poly(dA).poly(dT) insertion, whereas UV5 activity was not affected by this polymer. Neither the wt nor the UV5 promoter was influenced by the presence of poly(dG).poly(dC). Quantitation of abortive transcripts from supercoiled templates showed that the influence of the polymers on promoter activity was small. Comparative studies of the primary promoter with a secondary promoter (which initiates near -22), activated by 25% glycerol, were performed.

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. I. Cloning and characterization studies.

To assess the role of neighboring DNA sequences in gene regulation, poly(dA).poly(dT) and poly(dG).poly(dC) were cloned adjacent to promoters of the lactose control region. Recombinant plasmids were constructed which were suitable for large scale purification of restriction fragments containing these promoters, 95-base pair (bp) AluI fragments containing the lack operator and promoter for the lac wild type and for the catabolite gene activating the protein-independent mutant, lac UV5, were cloned into pBR322. Homopolymers of varying lengths were inserted into the -60 region of these promoters using recombinant DNA techniques. Six of the recombinant plasmids were chosen for detailed analysis: wild type (wt); wt-AT, containing 70 bp of poly(dA).poly(dT); wt-GC, containing 23 bp of poly(dG).poly(dC); UV5; UV5-AT, containing 70 bp of poly(dA).poly(dT) and finally UV5-GC, containing 43 bp of poly(dG).poly(dC). These plasmids were characterized by restriction mapping and DNA sequencing. The effects of the DNA homopolymers on the interaction of the Escherichia coli RNA polymerase with the promoters were studied using nitrocellulose filter binding. The results show that poly(dA).poly(dT) increases the level of RNA polymerase binding, whereas poly(dG).poly(dC) has no detectable effect.

Mutability of bacteriophage M13 by ultraviolet light: role of pyrimidine dimers.

The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag+ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.

Molecular mechanisms of substitution mutagenesis. An experimental test of the Watson-Crick and topal-fresco models of base mispairings.

The proteins coded by bacteriophate T4 replication genes 32, 41, 43, 44, 45, 61, and 62 together can replicate phi X174 DNA templates very efficiently. The fidelity of this in vitro replication reaction has been measured using an infectivity assay. The product molecules have the same specific infectivity as the template DNA. When an amber mutant DNA template is used, no increase in the frequency of revertants is seen even after more than 60 duplications in vitro. By using imbalances in the concentrations of deoxynucleotide substrates, the error rate during DNA replication in vitro can be greatly increased. Control experiments indicate that the increased mutagenesis is not due to the presence of dITP or dUTP as contaminants in the deoxynucleotide substrates used. The increase in the frequency of revertants is linearly related to the ratio of the correct and the incorrect deoxynucleotides. Determination of the DNA sequence of the revertants induced shows that a change in DNA sequence of the amber site predicted from the nucleotide bias occurs. DNA synthesis in vitro resembles in vivo replication in that the error rate depends not only upon the base change required for reversion but also upon the neighboring DNA sequences. The error rate is estimated to be 5 X 10(-6) at am3 site, 6.4 X 10(-7) at am86 site, and less than 2.9 X 10(-7) at am9 site. Comparison of the frequency of G-T and A-C mispairs reveals that most AT leads to GC transition mutations occur through G-T mispairs. Measurement of the frequency of the mispairs required to induce transversion mutations reveals that these occur primarily through purine-purine mispairs. Transition mutations are more frequent than transversion mutations at both the am3 and the am86 sites. These observations support the models for base pairing errors proposed by Watson and Crick ((1953) Nature 171, 964-967) and Topal and Fresco ((1976) Nature 263, 285-289).

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).