Intramolecular Heterogeneity Research Articles

Physicochemical characterization of mitochondrial DNA from pea leaves.

The mitochondrial DNA from pea leaves exists in a circular conformation. 25% of the circular molecules exist as supercoils, and 10% of the molecules are dimers. The molecular weight of mitochondrial DNA is about 66 to 70 x 10(6) by electron microscopy, and 74 x 10(6) from its renaturation kinetics. No evidence for inter- and intramolecular heterogeneity is found.

Role of base composition in the electrophoresis of microbial and crab DNA in polyacrylamide gels.

Intramolecular heterogeneity of eukaryotic nuclear DNA is shown by main-band and satellite DNAs. The function of the latter is uncertain, but they migrate more slowly in electrophoresis; this seems to be determined by base composition.

Intramolecular heterogeneity of yeast mitochondrial DNA

After sonication, yeast mitochondrial DNA analyzed on CsCl density gradient shows several components, of which some have a higher and others a lower buoyant density than that of the undergraded mitochondrial DNA (1.684 g/cm 3). Occurrence of different components was also observed after sonication of mitochondrial DNA prepared from a cytoplasmic respiratory-deficient mutant (DM 1). It was shown (1) that DM 1 mitochondrial DNA has a buoyant density of 1.671 g/cm 3 corresponding to A-T and G-C contents, respectively of 96% and 4%. The intramolecular heterogeneity observed after sonication suggests that the G-C content could not be uniformly distributed along the DNA molecule, but could be localized in a limited region of the molecule. If this is true, this DNA could still have genetic information.

Mature form of the deoxyribonucleic acid from chick embryo lethal orphan virus.

The deoxyribonucleic acid (DNA) of chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, had a biphasic denaturation profile indicating intramolecular base composition heterogeneity. This was confirmed by shearing the DNA and centrifuging it to equilibrium in Cs(2)SO(4) in the presence of HgCl(2) when two bands were formed. No circular molecules formed when CELO virus DNA was annealed, although lambda DNA formed circles under the same conditions. No circular molecules were found by sedimentation or electron microscopy when the DNA was digested with exonuclease III and then annealed, but 30 to 40% of T7 DNA molecules became circular under similar conditions. The complementary strands of CELO virus DNA both appeared to be continuous, and, when CELO DNA was denatured and then annealed under appropriate conditions, all of the renatured molecules were linear. It is concluded that CELO virus DNA consists of a unique rather than permuted collection of linear molecules that lack exposed single-strand complementary ends or duplex terminal repetitions. These results are discussed in relation to the replication of viral DNA and the transformation of host cells.

Denaturation and the renaturation kinetics of chloroplast DNA from Chlamydomonas reinhardi

The chloroplast DNA of Chlamydomonas was obtained by CsCl preparative centrifugation. Denaturation studies showed intramolecular heterogeneity. Renaturation kinetics revealed a major component of the DNA with a genomic size of about 2 × 10 8 daltons and a minor component, constituting about 10% of the total DNA, with a genomic size estimated at 1 to 10 × 10 6 daltons. The value of 2 × 10 8 daltons corresponds to about 24 copies of the genome per chloroplast. The relationship between this value and the diploidy shown by genetic analysis is discussed.

The intramolecular heterogeneity of Bacillus subtilis DNA studied by isopycnic CsCl gradient

The intramolecular heterogeneity of Bacillus subtilis DNA studied by isopycnic CsCl gradient

Intramolecular heterogeneity of the deoxyribonucleic acid of temperate bacteriophages.

Spectral changes accompanying the thermal denaturation of phage deoxyribonucleic acid suggested that lambda is not unique in possessing large-scale intramolecular heterogeneity and nucleotide clustering; instead, lambda seems to share this property with other enteric phages.

Spectral analysis of the intramolecular heterogeneity of λ DNA

The internal heterogeneity of base composition of DNA derived from λ bacteriophages has been detected by studies of the changes in ultraviolet absorption spectrum that occur during the course of thermal denaturation. Results obtained for the internal heterogeneity of λ c I-857 DNA are consistent with results obtained by shearing methods. Results obtained with deletion mutants of λ are consistent with the known locations of the deletions in the chromosome. In the case of the b 2 deletion, it appears possible to detect heterogeneity of base composition within the b 2 region. The method gives results which complement those obtained by shearing; it is also applicable to phage DNA of permuted base sequence, which cannot be studied by shearing methods.

The melting transition of low-molecular-weight DNA: Theory and experiment

One of the major difficulties in calculating transition curves for the “melting” of helical polynucleotides is to take account of the fact that all of the base pairs do not have the same stability, a point not considered in detail in the early theoretical treatments of the transition. In this paper it is shown that the resulting intra-molecular heterogeneity has a profound effect on calculated melting curves, and that transition curves in satisfactory agreement with experiment can be calculated only if the theory recognizes the difference between base pairs. Because of the mathematical complications which accompany this extension of the theory, a simplified model is used: it is assumed that the molecules are short enough for all of the bonded base pairs in a molecule to be clustered together, with unbonded bases only at the ends. This model is a good approximation when the molecular weight is below a certain upper limit, found in practice to be several hundred thousand. Two kinds of base pairs of differing stability, corresponding to adenine—thymine and guanine—cytosine, are recognized by the model used. It is found that the calculated transition curves depend on the sequence in which they are arranged; for the purpose of calculation a random sequence is assumed. Transition curves were obtained with computer random-sampling techniques. Experimental melting curves for a series of shear-degraded bacteriophage T2 DNA samples have been compared with theoretical curves, with good agreement at low molecular weights. Separation of the strands is taken into account, both in the theory and in the experiments.

Thermal dissociation of synthetic lambda (λ) RNA- Escherichia coli DNA hybrids

The possible existence of nucleotide sequence complementarity between the temperate phage lambda (λ) and its host, Escherichia coli, was examined by means of thermal dissociation studies of RNA-DNA hybrids. Radioactive λ RNA and T4 RNA were synthesized “in vitro” using the DNA-dependent RNA polymerase. RNA-DNA hybrids were formed between λ RNA and λ DNA, T4 RNA and T4 DNA, and λ RNA and E. coli DNA. The λ RNA-λ DNA hybrid dissociated over the same temperature range as the λ RNA- E. coli DNA hybrid. This range was considerably broader than that exhibited by the T4 RNA-T4 DNA complex. Correlation of the shape and average dissociation temperature ( T M ) 4 4 The following abbreviations will be used: T M = temperature at which half the RNA has dissociated from DNA; 1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate; A-T and G-C = adeninethymine and guanine-cytosine base pairs, respectively. of the melting curves of the RNA-DNA hybrids with those previously reported for λ DNA and T4 DNA provides physical evidence that nucleotide sequence complementarity exists between primer DNA and the synthetic RNA product. It is also possible to conclude that complementarity exists between λ DNA and E. coli DNA. The broad range of dissociation temperatures for the λ RNA-λ DNA and λ RNA- E. coli DNA hybrids demonstrates the presence of a relatively large amount of intramolecular chemical heterogeneity in λ DNA.

Evidence for intramolecular heterogeneity in pneumococcal DNA

Pneumococcal DNA of molecular weight about 107, bearing four independent transforming markers, was degraded by mechanical shearing to mol. wt. 6, 2 and 1 × 106 respectively. The ordering by density in a CsCl gradient of the four transforming activities depended upon the mol. wt. of the DNA. One marker, in particular, became more dense as the size of the fragments bearing it was reduced to 1 million mol. wt. Analytical bandings of fractions from the preparative band showed greater density differences within the lower mol. wt. DNA bands; other estimates show that the heterogeneity increased approximately as 1/√M. The markers also changed in heat stability as the mean mol. wt. of the DNA decreased, but with an entirely different apparent base composition order. Hence, base composition cannot be the sole controlling factor for both density and heat denaturation. It is concluded that irreversible collapse of DNA molecules at elevated temperatures is controlled by local fluctuations of composition within short regions and that there are wide ranges of composition over sufficiently large regions of the DNA to be of biological significance. The physical properties of a given transforming activity are controlled by the entire piece of DNA with which it is associated rather than merely with the local marker region itself.