Stability mapping along the DNA double strand and its relation to the genetic map.

The distribution of the stability of the double helical structure along the whole DNA of fdphage and its restriction fragments is calculated. In this calculation, Poland's method, which has been established as a rigorous algorithm for taking the base sequence explicitly into consideration, is used. The molecular thermodynamic parameters in the calculation have been determined so as to best reproduce the melting profile of the DNA and its fragments. The results, which are presented as melting maps, show fairly good agreement with those experimentally obtained by the present authors earlier. A close correlation with genes in the genetic map is apparent for some cooperatively melting regions observed in the stability map.     

Denaturation map of polyoma DNA.

A denaturation map of polyoma DNA cleaved by Eco R1 to form linear molecules was established by electron microscopy. Partial denaturation, under the same conditions, of fragments obtained by Haemophilus influenzae restriction enzymes allowed us to align the denaturation map with the already established physical map of polyoma DNA (Griffin et al., 1974).     

Differential scanning calorimetric and theoretical studies of ColE1 DNA

The differential scanning calorimetry (DSC) of plasmid ColE1 DNA was carried out. The DSC curve under the solvent condition of 1.0 X SSC buffer gave eleven clear peaks over the temperature range of 83 to 98 degrees C. The DSC curves obtained here were essentially in good agreement with the optical melting curves of ColE1 DNA reported previously. The theoretical melting profiles of ColE1 DNA calculated from its entire nucleotide sequence showed a good agreement with the DSC curves. The theoretical analysis made by constructing the thermal stability map showed that there was the positional correlation between the boundaries of the cooperatively melting regions and the ends of the protein coding regions of genes of ColE1. It was shown that the helix-coil transition of many of the small genes had a single cooperatively melting region. However, the large genes such as cea and mob3 had two or more cooperatively melting regions. It was suggested that this is closely related to the domain structures of the proteins encoded by such genes.     

鱼肝线粒体DNA的简易提纯方法

本文报道一种简易分离纯化线粒体DNA(mtDNA)的方法。即以差速离心或蔗糖梯度离心制得线粒体后,用RNase除去RNA,可得纯mtDNA,或用凝胶过滤、或低熔点琼脂糖法纯化之。所获纯mtDNA可用于限制性酶切图谱的组建和其片段的克隆。此法可避开冗长的昂贵的超速离心,故可为普通实验室所采用。文中还讨论了mtDNA纯化和得率的因素。     

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.     

Electron microscope maps of SA7 DNA melting

Electron microscopic denaturation maps corresponding to the first peaks of the differential melting curve of SA7 DNA were constructed by fixation of partly denatured molecules with glyoxal at temperatures within the melting range. These maps were oriented with respect to the functional map of the virus genome. The localization and the size of the most AT-rich SA7 DNA regions were determined.     

Physical and genetic analyses of IncI2 plasmid R721: evidence for the presence of shufflon

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.     

Variations in the satellite DNA content of Cucumis melo in relation to dedifferentiation and hormone concentration.

Total DNA from Cucumis melo contains a 1.706 satellite DNA which can be resolved into two components; one of these components has a higher temperature of melting (component I) then the other component II). In this study, we have further investigated these components by thermal denaturation and by Eco R1 digestion. Component I reveals a homogeneous melting profile and is only partially cleaved by Eco R1, whereas component II reveals a heterogeneous melting profile and is entirely digested by Eco R1. A possible mitochondrial origin for component II is discussed. When an in vitro culture of root tissues from Cucumis melo was initiated or when the phytohormone (NAA) concentration of established cultures was modified, a new satellite DNA (1.719) appears transitorily and the satellite DNAs already present in seedlings undergo quantitative and possibly qualitative variations. Satellite DNAs therefore seem to be involved in the response to some physiological variations.     

Characterization by molecular cloning of insertion mutants in TOL catabolic functions

A physical and genetic map of the Tol catabolic region of pWWO (TOL) was obtained by restriction endonuclease analysis of several DNA insertion mutants (xylA, xylA xylS, xylS, and xylR) of R plasmid--TOL derivatives. In two cases, the inserted DNA was shown from restriction, DNA hybridization, or heteroduplex analysis of cloned Hind III fragments to originate from within pWWO fragment Hind III-E. The effect of these DNA insertions on Tol catabolic activity and on structural alterations to the TOL plasmid is discussed.     

DNA of the Streptomyces phage SH10: Binding sites for Escherichia coli RNA polymerase and denaturation map

Summary Escherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.     

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.     

Electron microscopy of late adenovirus type 2 mRNA hybridized to double-stranded viral DNA.

R loops were generated with late adenovirus type 2 (Ad2) mRNA in double-stranded viral DNA, and visualized by electron microscopy. Unpaired DNA sequences in Ad2:Ad2+ND4 heteroduplex DNA served as a visual marker for the orientation of R loops with respect to the conventional DNA map. The most abundant classes of late Ad2 mRNA observed by this technique hybridized, in order of R-loop frequency, with midpoints near posit1ons 0.57, 0.88, 0.77, and 0.40 to 0.50 of the DNA map. The R loop at position 0.57, 0.88, 0.77, and 0.40 containing the hexon gene; the one at position 0.88 corresponded to a region containing the fiber gene. The relative frequencies of these two R loops paralleled those of the encoded gene products. The mRNA sizes, calculated from those of the respective R loops, were slightly larger than needed to code for these polypeptides. Using the R-loop technique, two locations at which adjacent mRNA''s hybridized to different strands were accurately mapped at positions 0.61 and 0.91 of the DNA. The map positions of late Ad2 mRNA correlated well to published RNA and protein maps.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.     

Melting fine structure of filamentous fungus nuclear DNA.

Melting fine structure of the nuclear DNA isolated from the filamentous fungus Fusarium graminearum Schwabe is presented. Optical melting profiles of nuclear DNA were analyzed by using a combination of curve fitting and derivative techniques. The "melting components" were obtained from the derivative curve by a simple decomposition technique. Differential optical melting curves of unsheared nuclear DNA indicate the presence of 15 "melting components" in filamentous fungus nuclear genome. It should be emphasized that the "melting components" observed here are different from the "thermalites" which can be observed in bacteriophage DNA. The "melting components" reported here represent the separately melting of large "blocks" of fungus nuclear DNA.     

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

tra cistrons and proteins encoded by the Escherichia coli antibiotic resistance plasmid R6-5

Summary Hybrid plasmids obtained by cloning individual EcoRI and HindIII fragments of the conjugative plasmid, R6-5, were analyzed for their ability to complement transfer-deficient point mutations of Flac. As a result, the locations of 10 tra cistrons were defined on the physical map of R6-5. Two cistrons, traE and traG, are interrupted by EcoRI restriction sites and one cistron, traC, probably contains a HindIII restriction site. The origin of DNA transfer, oriT, was also localized. Surprisingly the hybrid plasmid carrying oriT is mobilized by the F factor as well as by R6-5. The surface exclusion cistrons, traS and traT, were mapped and their biological expression analyzed. A total of 18 proteins encoded by cistrons within the tra region were detected by SDS polyacrylamide gel electrophoresis of proteins synthesized in minicells; they represent about 53% of the coding capacity of the cloned DNA. R6-5 DNA fragments containing the cistrons traC, traE, and traT directed the synthesis of proteins which comigrated during SDS gel electrophoresis with the F-coded proteins previously characterized as TraCp, TraEp, and TraTp. A further two proteins encoded by R6-5 comigrated with F-encoded (but genetically unidentified) proteins whose cistrons map in the corresponding part of the tra region. In contrast, no R6-5 proteins corresponding to F proteins TraAp, TraDp, TraJp, TraMp, 6a or 6c were detected. These results are discussed in relation to known DNA sequence homologies between the F and R6-5 plasmids. A preliminary physical map of the tra region of R6-5 is presented and compared with that of F.