Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis. Denaturation mapping by electron microscopy.

Electronmicroscopic observation of the denaturation pattern of 130 partially denaturated linear mitochondrial DNA molecules from Saccharomyces carlsbergensis was used to investigate the distribution of AT-rich sequences within the mitochondrial genome. The molecules were observed after heating to 43 degrees C in the presence of 12% formaldehyde. These conditions resulted in an average denaturation per molecule of 21%. The average length of the molecules was 10 mum, and a few molecules had a length corresponding to the size of the complete genome. The undenaturated regions varied in length from 0.1 to 5.0 mum with denaturated regions of length 0.02 to 0.1 mum in between. A denaturation map was constructed by use of one of the long molecules (28.7 mum) as a master molecule for positioning of all other molecules. This map shows distinct regions corresponding to the position of easily denaturated sequences in the mitochondrial DNA. These sequences which presumably correspond to the very AT-rich regions, known to exist in the yeast mitochondrial DNA, were found at intervals of about 0.5 - 3 mum on the map.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.     

Anatomy of herpes simplex virus DNA. V. Terminally repetitive sequences.

Native DNA from four strains of herpes simplex virus 1 (HSV-1) circularized after digestion with the lambda exonuclease, indicating that the molecules were terminally repetitious. In two strains, the terminal repetition was evident in nearly 50% of the DNA molecules. Maximal circularization was observed when only 0.25 to 0.5% of the DNA was depolymerized by the exonuclease, suggesting that the minimal size of the terminally repetitious regions is in the range of 400 to 800 bases pairs. More extensive exonuclease treatment resulted in a reduction in the frequency of circularization. To determine whether the terminally repetitive regions themselves contained self-annealing sequences that were precluding circularization of more extensively digested DNA, the terminal fragments from HinIII restriction endonuclease digests were isolated, denatured, and tested for their ability to self-anneal. The results of hydroxyapatite column chromatography and electron microscope examination of the terminal regions are consistent with this hypothesis.     

Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields.

Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

Natural occurrence of left-handed (Z) regions in PM2 DNA

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.     

Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.     

Natural Occurrence of Left-Handed (Z) Regions in PM2 DNA

Abstract

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05–0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.     

The high affinity binding site on polyoma virus DNA for the viral large-T protein.

In order to map the high affinity binding site for the viral large-T protein on polyoma virus DNA, we have developed an assay which does not require purified protein. It is based on the specific elution of the large-T ATPase activity from calf thymus DNA cellulose by recombinant DNA molecules including known sequences of the viral DNA. Using this assay, a high affinity binding site has been mapped on the early region side of the ori region. Binding requires the integrity of a sequence /AGAGGC/TTCC/AGAGGC/ (nucleotides 49 to 64 in the DNA sequence of the A2 strain). Similar repeats of a PuGPuGGC sequence within less than 20 bases are not found within the viral coding regions, but are strikingly common in the control regions of papovaviruses and other eukaryotic DNAs.     

Unique translational positioning of nucleosomes on synthetic DNAs.

A computational study was previously carried out to analyze DNA sequences that are known to position histone octamers at single translational sites. A conserved pattern of intrinsic DNA curvature was uncovered that was proposed to direct the formation of nucleosomes to unique positions. The pattern consists of two regions of curved DNA separated by preferred lengths of non-curved DNA. In the present study, 11 synthetic DNAs were constructed which contain two regions of curved DNA of the form [(A5.T5)(G/C)5]4 separated by non-curved regions of variable length. Translational mapping experiments of in vitro reconstituted mononucleosomes using exonuclease III, micrococcal nuclease and restriction enzymes demonstrated that two of the fragments positioned nucleosomes at a single site while the remaining fragments positioned octamers at multiple sites spaced at 10 base intervals. The synthetic molecules that positioned nucleosomes at a single site contain non-curved central regions of the same lengths that were seen in natural nucleosome positioning sequences. Hydroxyl radical and DNase I digests of the synthetic DNAs in reconstituted nucleosomes showed that the synthetic curved element on one side of the nucleosomal dyad assumed a rotational orientation where narrow minor grooves of the A-tracts faced the histone surface with all molecules. In contrast, the curved element on the other side of the nucleosome displayed variable rotational orientations between molecules which appeared to be related to the positioning effect. These results suggest that asymmetry between the two halves of nucleosomal DNA may facilitate translational positioning.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Automatic construction of restriction site maps.

A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.     

The relation of single-stranded regions in bacteriophage PM2 supercoiled DNA to the early melting sequences.

Bacteriophage PM2 supercoiled DNA contains one to three small single-stranded regions that can be detected in the electron microscope after various treatments. The relative positions of these regions were mapped against the unique cleavage site for the restriction endonuclease R · HapII on PM2 DNA. Any of eight sharply defined regions of the genome may be single-stranded in supercoiled molecules. They are found in all possible combinations of three or less and at approximately the same frequency. A comparison of this map of supercoiled DNA with the alkaline denaturation pattern of nicked circular or linear PM2 DNA showed that these same regions were also the earliest melting regions in non-supercoiled DNA.     

Dehydrating agents sharply reduce curvature in DNAs containing A tracts.

The structural basis of DNA curvature remains elusive, because models for curvature based on crystallographic structures of molecules containing A tracts do not agree with any of the models for sequence-directed curvature based on solution studies. Here we demonstrate that the difference is probably due to MPD (2-methyl-2,4-pentanediol), the dehydrating agent commonly used in crystallography. One characteristic signature of curved DNA molecules is that they run anomalously slowly on polyacrylamide gels, appearing to be larger than they actually are. The gel anomalies of three curved DNAs from trypanosome kinetoplast minicircles drop monotonically with increasing MPD concentration, indicating that MPD straightens molecules that are curved in aqueous solution. This is not due to some non-specific effect of MPD on poly(dA) or polypurine tracts, because control molecules containing dA70 and dG43 run normally over the full range of MPD concentrations. Circular dichroism spectra are not affected by MPD, ruling out a conformational change to a structure outside the B-DNA family. The effect is not due to MPD-induced changes in phasing of the curved sequences, because MPD has virtually no effect on the linking numbers of relaxed plasmids containing either curved sequences or dA70. At the concentrations of MPD used in X-ray crystallography, the curvature of DNAs containing A tracts is substantially lower than in solution, which probably explains the ongoing discrepancies between the crystallographic results and models based on solution studies.