Bending and flexibility of kinetoplast DNA

We have evaluated the extent of bending at an anomalous locus in DNA restriction fragments from the kinetoplast body of Leishmania tarentolae using transient electric dichroism to measure the rate of rotational diffusion of DNA fragments in solution. We compare the rate of rotational diffusion of two fragments identical in sequence except for circular permutation, which places the bend near the center in one case and near one end of the molecule in the other. Hydrodynamic theory was used to conclude that the observed 20% difference in rotational relaxation times is a consequence of an overall average bending angle of 84 +/- 6 degrees between the end segments of the fragment that contains the bending locus near its center. If it is assumed that bending results from structural dislocations at the junctions between oligo(dA).oligo(dT) tracts and adjacent segments of B DNA, a bend angle of 9 +/- 0.5 degrees at each junction is required to explain the observations. The extent of bending is little affected by ionic conditions and is weakly dependent on temperature. Comparison of one of the anomalous fragments with an electrophoretically normal control fragment leads to the conclusion that they differ measurably in apparent stiffness, consistent with a significantly increased persistence length or contour length in the kinetoplast fragments.     

Physical characterization of a kinetoplast DNA fragment with unusual properties

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.     

DNA sequence analysis: a formula to predict electrophoretic mobilities of oligonucleotides on cellulose acetate

A simple method has recently become available for sequence analysis of large oligonucleotide fragments. Sequences are derived from the characteristic mobility shifts of the sequential partial degradation products of the oligonucleotide on two dimensional homochromatography. We have now developed an empirical formula for predicting the relative mobilities of each of the partial products in the first dimension (electrophoresis on cellulose acetate gel). The formula allows a more precise interpretation of the sequence of the oligonucleotide. It eliminates the ambiguities present in the method previously reported for sequence analysis by simple inspection of the mobility shifts. In order to amplify the mobility shifts so that they may be more easily and accurately measured, methods have been developed for preparing and fractionating the oligonucleotides on 40 × 40 cm DEAE-cellulose plates. Both improvements have proven valuable for direct sequence analysis by mapping.     

A highly bent fragment of Crithidia fasciculata kinetoplast DNA

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.     

Effects of sequence selective drugs on the gel mobility of a bent DNA fragment.

The effects of various drugs on the structure of a bent DNA fragment have been investigated by studying DNA mobility in polyacrylamide gels. This DNA fragment has an anomalously slow rate of migration on account of its phased runs of adenines. Nogalamycin and echinomycin increase the gel mobility of kinetoplast DNA suggesting that the bending has been removed. Mithramycin, actinomycin, distamycin and ethidium have either no effect or cause a further reduction in mobility. These results are compared with other, non-bent DNA species which always show a decrease in gel mobility in the presence of DNA binding drugs.     

DNA fragments with specific nucleotide sequences in their single-stranded termini exhibit unusual electrophoretic mobilities.

DNA restriction fragments, 120-650 base pairs (bp) in length, with 5'-GCGC-3', 5'-GGCC-3' or 3'-GCGC-5' single-stranded overhanging termini, give rise to diffuse bands of unusual electrophoretic mobility in non-denaturing polyacrylamide gels. This shift in electrophoretic mobility can be observed at 4-12 degreesC, not at higher temperatures, but is stabilized by 5-10 mM Mg2+, even at 37 degreesC. The nucleotide sequence in the abutting double-stranded part of the fragment does not affect this phenomenon, which is not caused by dimerization. The altered mobility may be due to the unusual terminal DNA structure, which is dependent on co-operative interactions among more than two neighboring G and C residues. The structure is stabilized by cytidine methylation. The biological role of such fragment structures in DNA repair and recombination is presently unknown.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

On the flexibility and the dynamics of DNA

We describe and discuss the stability conditions of a naked double stranded DNA molecule starting from the evaluation of condensation or compactness fluctuation of this molecule embedded in pure water.     

Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.     

Temperature dependence of the gel electrophoretic mobility of superhelical DNA.

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a function of both temperature and of linking number (Lk). At temperatures below 37 degrees, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37 degrees the electrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37 degrees - 65 degrees the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37 degrees and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6 degrees, which lies within 3 degrees of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.     

Protein and DNA sequence determinants of thermophilic adaptation

There have been considerable attempts in the past to relate phenotypic trait—habitat temperature of organisms—to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT) of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from −10 °C to 110 °C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL), and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = −0.10). On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.     

Transblot studies with biotin-labeled proteins: electrophoretic mobilities and detection limits

Molecular weight standard proteins, mouse IgG as well as several antigens cross-reacting with the carcinoembryonic antigen (CEA), were biotin labeled, submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The bound proteins were revealed by the use of avidin-peroxidase conjugates and a suitable substrate. The ratio of N-hydrosuccinimido biotin (NHSB) to protein yielding the lowest detection limit was determined. At an optimal NHSB/protein ratio, 0.33 ng of IgG heavy chains and 0.17 ng of IgG light chains could be visualized. With the exception of human albumin and ovalbumin, the increase in apparent molecular weight after biotin labeling was less than 10% for the proteins tested. The method has proven to be a valuable addition to Western blots performed with CEA-related antigens and monoclonal anti-CEA antibodies.     

Nucleotide sequence of a reiterated rat DNA fragment

A reiterated component of rat DNA was isolated by restriction with HindIII endonuclease and polyacrylamide gel electrophoresis. Sequence analysis revealed that the fragment was 179 nucleotides long. Unlike the known 370N reiterated rat DNA fragment which contained a high m5C content (2.7 mole%), this repetitive element contained a rather low m5C content (0.5 mole%). The present rat repetitive sequence appeared to be of alpha-type as shown by its significant homologies with alpha DNA sequences of African green monkey and human. The possibility of sequence heterogeneity is discussed.     

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.