High resolution thermal denaturation analyses of small sequenced DNA restriction fragments containing Escherichia coli lactose genetic control loci.

Differential melting curves are reported for four DNA restriction fragments (789, 301, 203, and 95 base pairs in length) spanning the lactose control region. All but the smallest melt with two or more subtransitions. Maps are proposed which identify the positions of regions of different thermal stability in the sequences. The sizes of regions comprising subtransitions range from 60 to 200 base pairs. An analysis is made of the cooperativity exhibited between regions in the sequence. The effect on the shape of the differential melting curves of Na+ between 10 mM and 0.5 M as well as that of Mg2+ and glycerol has been determined. An 81-bp-long sequence of unusual thermal stability occurs at the lactose promoter. Its TM change, resulting from the above change in salt concentration, is out of step by 1.5 degree C with the neighboring DNA sequence. The potential biological significance of this behavior is discussed.     

High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region

The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence. The thermal denaturation of these fragments was measured at 3 salt concentrations. The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed. The existence of two thermodynamic boundaries in this region is also confirmed. The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm. The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration. The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.     

Nucleotide sequence analysis of a mouse Y chromosomal DNA fragment containing Bkm and LINE elements

The strong suppression of crossing-over between the X and Y chromosomes permits rapid accumulation of repetitive sequences in the Y chromosome. To gain insight into the mechanism responsible for the sequence amplification, it is essential to characterize Y chromosomal repetitive sequences at the molecular level. Here, we report the entire nucleotide sequence (3,902bp) of AC11, a mouse sequence that is repeated 300 times in the Y chromosome. AC11 is AT rich (32.8% GC), and contains many short poly(A) sequences. In addition, it has Bkm and LINE sequences as well as a Y chromosome-specific sequence. The Bkm sequence consists of typical (GATA) and (GACA) repeating units, whereas the LINE sequence deviates considerably from other mouse LINE sequences (71–76% identity) and may be considered atypical. The Y chromosome-specific region seems to be unique and does not identify similar sequences in the GenBank library. The information obtained from the nucleotide sequence should form the foundation to study the evolutionary processes through which AC11-related sequences have accumulated in the mouse Y chromosome.     

Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )     

High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions.

The fidelity achieved in first derivative profiles of DNA thermal denaturation is shown to depend on a number of factors including the thermal increment of data gathering, the precision of absorbance readings, and the manner in which data are smoothed prior to calculating the derivative of hyperchromicity. The closeness with which thermal denaturation data can be fitted by a cubic polynomial is carefully considered, and a derivation is presented for the estimated error in calculated values of the derivative of hyperchromicity with respect to temperature. After reviewing both theoretical and experimental evidence for the expected minimum width of a thermal transition in DNA, we conclude that thermal increments of 0.05°C or less are required for an adequate representation of transitions in naturally occurring DNA's. Data gathered under conditions meeting the requirements suggested here for quantitative recording of thermal denaturation profiles (Vizard and Ansevin, submitted for publication) show that virtually all of the high-resolution thermal denaturation profile of a simple, naturally occuring DNA may consist of small subtransitions, which we call thermalites. The finding of substransitions is consistent with current theories of DNA melting. A particularly well-resolved thermalite of λ bacteriophage DNA has a breadth of only 0.30°C (2σ width), and thus is narrower than previously reported thermal transitions for DNA. For this thermalite, the combination of width, shape, and position in the profile suggests that the substransitions observed in accurately recorded DNA thermal denaturation profiles are not described satisfactorily by existing theories. Knowledge of the requirements for the quantitative recording of thermal denaturation profiles should greatly favor the usefulness of denaturation experiments for physical genomic analysis.     

Comparison of the chemical and thermal denaturation of proteins by a two-state transition model

The conformational stabilities of eight proteins in terms of the free energy differences between the native "folded" state of the protein and its "unfolded" state were determined at 298 K by two methods: chemical denaturation at 298 K and extrapolation to 298 K of the thermal denaturation results at high temperature. The proteins were expressed in Escherichia coli from the Haemophilus influenzae and E. coli genes at different levels of expression, covered a molecular mass range from 13 to 37 kg mol(-1) per monomeric unit (some exhibiting unique structural features), and were oligomeric up to four subunits. The free energy differences were determined by application of a two-state transition model to the chemical and thermal denaturation results, ranged from 9.4 to 148 kJ mol(-1) at 298 K, and were found to be within the experimental uncertainties of both methods for all of the proteins. Any contributions from intermediate states detectable from chemical and thermal denaturation differences in the unfolding free energy differences in these proteins are within the experimental uncertainties of both methods.     

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

Summary The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.     

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.     

Comparison of the thermal denaturation behaviour of DNA-solutions and metaphase chromosome preparations in suspension.

Hyperchromicity measurements are well established to analyse the thermal denaturation behaviour of pure DNA sequences in solution. Here, we show that under appropriate experimental conditions this technique can also be applied to study thermally controlled conformation changes of higher order DNA-protein complexes as for instance metaphase chromosome preparations in suspension. A computer controlled sensitive, upright double beam photometer with a heatable cuvette was constructed. Measurements of the temperature dependent extinction of both, solutions and particle suspensions are possible, since sedimentation effects of particles can be neglected due to the vertical optical axis in the probe cuvette. Thermal denaturation of metaphase chromosome preparations of human and Chinese hamster cells was investigated and compared to melting profiles of DNA solutions for two excitation wavelengths, 256 and 313 nm. The influence of neutral and low pH was considered. The results indicate that metaphase chromosome preparations show a thermal denaturation behaviour different from pure DNA. Whereas DNA solutions showed one pH dependent melting peak at 256 nm only, the peak pattern of metaphase chromosome preparations showed a large variability both at 256 and 313 nm. At neutral pH, in two temperature regions (40-55 degrees C and 75-82 degrees C) peaks were found indicating chromosome typical conformation changes independently from the mammalian cell species (Chinese hamster, human). In contrast to pure DNA, no typical reduction in the temperatures of peak maxima with decreasing pH was found for metaphase chromosome preparations of both cell types. These results may be relevant for further systematic studies of efficient thermal probe/target denaturation procedures in non enzymatic DNA-chromosome in situ hybridisation.     

Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases. SmaI, HinfI and HhaI, provided one or two different bands between two species, although the hybridization patterns generated with the others. HindIII, XbaI, StyI and HaeIII, were the same in both. RFLPs in the digested profiles with SmaI, HinfI and HhaI could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

The sequence of a 1.9 kb Bam x Hind III fragment from yeast has been determined. This fragment is part of a yeast 6.7 kb Hind III segment cloned into pBR322 (pY20). The fragment carries a single gene for a glutamate tRNA which has no intron. According to genetic analyses [1] this fragment also contains a yeast chromosomal replicator. We have analyzed the sequence for potential open reading frames and for several structural features which are thought to be involved in the initiation of DNA replication. Hybridization studies have revealed that portions of this sequence are repeated within the yeast genome.     

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes.

A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.