Partial Denaturation Mapping Research Articles

The sequence arrangement of drosophila melanogaster 5S DNA cloned in recombinant plasmids

Segments of Drosophila melanogaster DNA containing 5S rRNA genes have been propagated in recombinant plasmids using E. coli as a host and Col E1 as a vector. Electron microscope partial denaturation mapping, mapping by ferritin labeling and restriction enzyme-gel electrophoresis analysis all indicate that the Drosophila DNA inserts of these plasmids consist of tandem repeats of 5S genes and spacer regions. The repeat length is approximately 380 nucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer of length 260 ntp. The insert in one plasmid (pCIT9) consists of 32 contiguous repeats. Restriction enzyme-gel electrophoresis analysis shows that all these repeats have the same length within ± 5 nucleotides. This repeat length is estimated as 370 ± 20 ntp by gel electrophoresis and 390 ± 40 ntp by partial denaturation mapping. A second plasmid (pCIT19) contains three complete genes, two complete spacers and incomplete flanking spacer sequences. The two complete repeat units released by suitable restriction endonuclease digestions differ in length by 20 ± 5 ntp, with estimated lengths of 370 and 390 ntp. The positions and spacings of the genes on this plasmid have been observed directly by ferritin labeling and by partial denaturation mapping. The A+T content of the 5S DNA spacer region is calculated to be 68%. By in situ hybridization, cRNA transcribed from one plasmid hybridizes to polytene chromosomes only at band 56F, the known locus of the 5S rRNA genes. Spontaneous excision of some of the tandem repeat units from the recombinant plasmids occurs during growth in E. coli; the frequency of excision does not depend upon the recA character of the host, but is greatly increased by chloramphenicol treatment.

Site-specific recombination in bacteriophage lambda

Lambda attB-attP is a derivative of bacteriophage lambda that contains both attB and attP, two sites required for integrative recombination. Lambda attB-attP can undergo int-mediated recombination to yield progeny phages whose DNA is 15% shorter than that of the parental phage. We have studied intracellular phage DNA from an infection of lysogenic bacteria with λ attB-attP in the presence of int gene product, rifampicin and chloramphenicol. The majority of the intracellular phage DNA consisted of circles with lengths of 17.51, 15.09 and 2.38 μm. Partial denaturation mapping confirmed that the 15.09 and the 2.38-μm molecules arose by an int-mediated intramolecular recombination reaction of the type predicted by the Campbell (1962) model. A minor proportion of the circles (3%) were much larger (33.9, 30.2 and 4.7 μm); in these cases denaturation mapping indicated that both intra- and intermoleeular recombination could take place.

Episomal Viral DNA in a Herpesvirus saimiri -Transformed Lymphoid Cell Line

The lymphoid cell line #1670 has been derived from the infiltrated spleen of a tumor-bearing marmoset monkey infected with Herpesvirus saimiri. The cells contain both types of H. saimiri DNA, unique light (L-) DNA (36% cytosine plus guanine) and repetitive heavy (H-) DNA (71% cytosine plus guanine), without producing infectious virus. Viral DNA was found to persist in these cells as nonintegrated circular DNA molecules. Closed circular superhelical viral DNA molecules were isolated by three subsequent centrifugation steps: (i) isopycnic centrifugation in CsCl, (ii) sedimentation through glycerol gradients, and (iii) equilibrium centrifugation in CsCl-ethidium bromide. The isolated circles had a molecular weight of 131.5 +/- 3.6 x 10(6). This is significantly higher than the molecular weight of linear DNA molecules isolated from purified H. saimiri virions (about 100 x 10(6)). Partial denaturation mapping of circular molecules from #1670 lymphoid cells showed uniform arrangement of H- and L-DNA sequences in all circles. All denatured molecules contained two L-DNA regions (molecular weights of 54.0 +/- 1.8 x 10(6) and 31.5 +/- 1.3 x 10(6)) and two H-DNA regions (molecular weight of 25.6 +/- 1.9 x 10(6) and 20.0 +/- 0.8 x 10(6)) of constant length. Maps of both L-regions suggested that the sequences of the shorter L-DNA region were a subset of those of the longer region. The sequences of both L-regions had the same orientation. Circular molecules from H. saimiri-transformed lymphoid cell line #1670 appeared to represent defective genomes, containing only 75% of the genetic information present in L-DNA of H. saimiri virions.

Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster.

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: generation of small plasmids after integration

We have observed that integration of the R plasmid R100.1 into the chromosome of Escherichia coli is associated with the formation of small, covalently closed circular elements. Contour length measurements, partial denaturation mapping, and analysis of the deoxyribonucleic acid fragments produced by digestion of one of these, pLC1, with the restriction endonuclease EcoRI indicate that it is the r-determinant element of R100.1.

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.

Mitochondrial DNA replication in Paramecium aurelia. Cross-linking of the initiation end

Mitochondrial DNA from Paramecium aurelia consists of three types of molecules: linear monomers, linear dimers and lariats (circles with tails). These have been characterized by partial denaturation mapping and snapback reassociation after denaturation. The partial denaturation pattern of the linear monomers was highly asymmetric with one-half of the molecule more resistant to denaturation than the other. Similar mapping of the lariats and dimers indicated that replication originates at or very close to the more resistant end of the molecule and proceeds unidirectionally towards the other. This process results in the formation of linear dimers in which the two monomer units are associated in a head-to-head arrangement. The processing of DNA in this manner requires a strong linkage at the initiation end of the DNA which is maintained throughout the replication process. This linkage appears to be covalent by two criteria. In one-third of the monomers measured, a small denaturation bubble was observed at the end of the resistant half of the molecule. This denaturation bubble was also observed in a large proportion of the lariats and dimers. In the lariats, the bubble appeared directly opposite the fork in the circular portion of the molecule while in the dimers it appeared at the center of the molecule. In addition, the dimers, lariats and a significant proportion of the monomers exhibited a rapid, intrastrand snapback reassociation to double-stranded molecules following denaturation by heat. The linkage was resistant to Pronase, alkali and ribonuclease H but was cleaved in the monomers by S1 nuclease.

Partial denaturation mapping of cloned histone DNA from the sea urchin Psammechinus miliaris.

A cloned 5.6-kb DNA repeat unit of Psammechinus miliaris containing all five histone coding sequences of known order and polarity has been shown to fall clearly into a low melting and a high melting half by thermal denaturation. The topoligies of the DNA sequences thus defined were determined by partial denaturation mapping in the electron microscope to gain further insight into the distribution of spacers and genes within histone DNA.

A Partial Denaturation Map of Herpes Simplex Virus Type 1 DNA: Evidence for Inversions of the Unique DNA Regions

Partial denaturation maps of 30 HSV-I DNA molecules have been obtained using a procedure designed to avoid possible hydrolysis of the DNA at alkalilabile bonds. From the denaturation pattern of the long unique DNA region these molecules were divided into two groups comprised of 16 and 14 molecules. Histogram plots relating the precentage denaturation to position on the DNA for these two groups were aligned in a manner appropriate to the HSV-I genome model. It was apparent that these groups had the orientation of the long region inverted with respect to each other. Similarly, from the denaturation maps of the short unique region, the molecules were divided into two groups each comprising 15 molecules. Alignment of the histogram plots of these groups indicated that the orientation of the short region was inverted in one group relative to the other. These partial denaturation data confirm the presence of four HSV-I genome arrangements resulting from the possible combinations of inversions of the two unique DNA regions.

The macronuclear ribosomal DNA of Tetrahymena pyriformis is a palindrome

The macronuclear ribosomal DNA of Tetrahymena pyriformis (strain BVII, syngen 1) is extraohromosomal. The majority of the molecules are linear with measured mean molecular weights of 13.0 × 10 6 to 13.8 × 10 6 in various experiments. Several lines of evidence indicated that the double-stranded rDNA molecule is a palindrome in the terminology of Wilson & Thomas (1974). That is, taking into account the polarity of the polynucleotide strands, the sequence is the same read from either end of the molecule. The rDNA molecules had a partial denaturation map that was symmetrical about the center of the molecule. There were two sites susceptible to cleavage by the restriction enzyme EcoRI. Partial denaturation of the fragments indicated that these two sites were equidistant from the center of the rDNA molecule. There were two different sites susceptible to cleavage by the restriction enzyme Bam, which were also equidistant from the center of the rDNA molecule. The two polynucleotide strands of a palindromic DNA molecule are similar or identical and each is self-complementary. Thus each strand will rapidly renature with first-order kinetics following denaturation. Thermal denaturation of the rDNA of Tetrahymena revealed a complex melting curve with at least six transitions. When the temperature was reduced under conditions such that intermolecular reassociation was not expected to occur, the rDNA abruptly lost 35% of its hyperchromicity. When the products of this snapback renaturation were examined in the electron microscope, many of the molecules observed were double stranded, and had a contour length half the length of the native rDNA. Thermal denaturation of the snapback molecules yielded the same transitions as the original melt, but in different proportions, as predicted from the partial denaturation map. There was little or no mismatching in the self-complementary strands that could be detected by comparison of melting temperature ( t m) between the melt and the remelt. Cruciform structures were generated by incubating the rDNA in vitro. Although the rDNA of the amicronucleate Tetrahymena strain GL is slightly different in size and buoyant density, and in its partial denaturation map from that of strain BVII, it too has a palindromic structure.

A map of the sites on bacteriophage PM2 DNA for the restriction endonucleases HindIII and HpaII

The map of the seven sites for the restriction endonuclease HindIII ‡ ‡ The nomenclature used for restriction endonucleases suggested by Smith & Nathans (1973) is endo R. HindIII, a restriction endonuclease from Hemophilus influenza; endo R. HpaII, a restriction endonuclease from H. parainfluenza. These have been further abbreviated to HindIII and HpaII in the interests of readability. and the single site for endo R. HpaII on PM2 DNA was determined. This map was oriented with respect to the denaturation map of this DNA (Brack et al., 1975) by partial denaturation mapping of the fragments. A new method for localizing restriction fragments by DNA-DNA hybridization and electron microscopy is described.

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.

EcoRI endonuclease cleavage map of bacteriophage P4-DNA

Satellite bacteriophage P4-DNA is cleaved by EcoRI endonuclease at three specific sites, 0.702, 0.737, and 0.985 fractional lengths as measured from the left end of the molecule. The EcoRI cleavage map was determined by electron microscopic length measurements and agarose-gel electrophoresis. The cleavage map was oriented relative to the P4 partial denaturation map where the most readily denatured end is defined as the right end of the molecule. It was observed that the EcoRI cleavage sites on P4-DNA were not cleaved with equal efficiency.

Structure of herpes simplex virus DNA: Topography of the molecule: II. Partial denaturation map

A partial denaturation map of herpes simplex virus type 1 DNA was constructed after treatment of the DNA at pH 11.3. The map was asymmetrical, showing preferential strand separation at the following fractional lengths: 0.01, 0.04, 0.16, 0.23, 0.28, 0.54, 0.56, 0.57, 0.62, 0.66, 0.68, 0.75, 0.87, 0.91, 0.95, 0.96 and 0.99. After 90-min denaturation, single-stranded interruptions were detected within denatured parts of molecules.

Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.

Studies on the mechanism of replication of adenovirus DNA: III. Electron microscopy of replicating DNA

Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied during synchronized synthesis in the first round of replication after release of hydroxyurea inhibition. The branched intermediates represented early and the unbranched intermediates late stages in the replication cycle. Digestion of the branched intermediates with Eco RI endonuclease revealed that replication had started at the molecular right end (the A-T-rich end). This was confirmed by partial denaturation mapping of branched intermediates. These results confirm and extend a tentative model on the mechanism of Ad5 DNA replication.

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions ( g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.

Round of Replication Mutant of a Drug Resistance Factor

A derivative of the R factor NR1 (called R12) has been isolated which undergoes an increased number of rounds of replication each division cycle in Proteus mirabilis, Escherichia coli, and Salmonella typhimurium. The alteration resulting in the increased number of copies (round of replication mutation) is associated with the transfer factor component of the R factor. R12 has the same drug resistance pattern as NR1, is the same size as shown by sedimentation in a sucrose gradient and electron microscopy (63 x 10(6) daltons), and has the same partial denaturation map. The level of the R factor gene product chloramphenicol acetyltransferase has been examined in P. mirabilis and was found to be consistent with gene dosage effects. The plasmid to chromosomal deoxyribonucleic acid ratio of NR1 increases several fold after entry into stationary phase, whereas this ratio for R12 remains approximately constant. Individual copies of R12 are selected at random for replication from a multicopy plasmid pool. A smaller percentage of R12 copies replicate during amino acid starvation than has previously been found for NR1 in similar experiments.

Alignment of partial denaturation maps of circularly permuted DNA by computer

Alignment of partial denaturation maps of circularly permuted DNA by computer

Non-random circular permutation of phage P22 DNA

Phage P22 is known to have a linear duplex chromosome that is circularly permuted and terminally repeated. We have found, by constructing a partial denaturation map of mature P22 DNA, that circular permutation in P22 DNA is restricted: all of the ends of the mature DNA fall within 20% of each other on the physical map. The limited distribution of ends can be explained by the “headful” packaging model of Streisinger et al. (1967), with the additional specifications that: 1. (a) the intracellular precursor DNA is no longer than ten times the length of mature phage DNA; 2. (b) encapsulation of DNA starts at a unique site; 3. (c) encapsulation proceeds sequentially therefrom. This model is supported by the denaturation maps of two deletion phage DNAs. We found, as expected from our model, that the extent of permutation is a direct function of the length of terminal repetition. An independent demonstration of this relation between permutation and terminal repetition was done by denaturation-self-reannealing experiments using wild type and deletion phage DNAs. In the case of phage with large terminal repetitions (16%), we can discern discrete classes of molecules (using denaturation mapping), which we interpret as the first, second, etc. headful cut from the intracellular DNA concatemer.