George G. Markov

Hydroxyapatite column chromatography in procedures for isolation of purified DNA

Abstract Two procedures are described for the isolation of purified DNA from mammalian tissues by using hydroxyapatite chromatography. They avoid enzyme treatment and are easily carried out in one day. The first one is a modification of the MUP (8 m urea-0.24 m sodium phosphate buffer) method of Britten et al. (2) in which some technical difficulties (clogging of the column) are overcome and the yield and the purity of the DNA are improved. The second procedure represents a combination of the classical methods of lysis of muclei with hydroxyapatite chromatography of the DNA and may be especially convenient for the isolation of high-molecular-weight DNA.

Substances interfering with spectrophotometric estimation of nucleic acids and their elimination by the two-wavelength method

Abstract The uniformity of the optical properties of substances interfering with the spectrophotometric determination of nucleic acids in different tissues has been proved. The absorption spectra of these substances show that the chief conflicting component is most probably tyrosine. The application of the two-wavelength method is therefore very convenient for nucleic acid estimations in animal tissues. The two wavelengths at which the optical density should be measured are 260 mμ and 286 mμ for RNA and 268 mμ and 284 mμ for DNA. A comparison of results obtained by different methods is presented.

Heterogeneity of Streptomyces DNA.

Reassociation kinetic analysis of DNA led to the discovery of some important differences between prokaryotic and eukaryotic DNA. Ten years ago it was found that some of the nucleotide sequences of eukaryotic DNAs were repeated several tens to a million times [ 11. Repetitive sequences in prokaryotic DNAs have not been found by the conventional methods. Bacause of the second-order kinetics of reassociation of the prokaryotic DNAs they were considered as consisting of non-repeated sequences only. Until recently it was thought that the ribosomal RNA genes are the only repeated DNA sequences in prokaryotes (see [2]). Chiscon and Kohne [3] revealed a small fraction of repeated DNA in Escherichia coli cells and presented evidence for its extrachromosomal (plasmid) origin. Recently, a fast renaturing fraction in E. coli DNA representing 2% the whole genome was described [4,5]. Basing on the properties of this fraction the authors exclude its plasmid origin. These data give reason to assume that repetitive sequences may also exist in the genome of some other prokaryotes.

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.

On the heterogeneity of the slow reassociating (“unique”) DNA

SummaryThe slow reassociating fraction of mouse DNA (“unique DNA”), when allowed to reassociate in 0.14 m sodium phosphate buffer at 50 °C showed a biphasic melting curve with a transition at 78–80 °C. On the basis of this feature, the slow reassociating DNA was separated preparatively into two fractions: “unique DNA” I and II. Their duplexes showed differences with respect to thermal stability, S1 nuclease resistance and rate of reassociation. About one third of the sequences in each fraction were fraction-specific. The conclusion was drawn that for “unique DNA” I these should be the low repetitive or single copy related sequences (multigene families) and for “unique DNA” II—the unrelated single copy sequences or recent families of low repetitive not yet diverged sequences.

Fractionation of chromatin by thermal precipitation in phosphate buffer

Abstract The stability of sonicated rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied as a function of buffer concentration (0.012 to 0.16 m ) and temperature (20 to 98 °C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 50 °C, 70 to 75 °C and above 90 °C) and two transitional zones (55 to 70 °C and 75 to 90 °C) on the A 320 curves and on the percentage precipitated nucleoprotein versus temperature curves as well. This permitted the fractionation of chromatin in 0.08 m -phosphate buffer into three fractions by a stepwise heating at 50 °C (50 °C-pellet) and 98 °C (50–98 °C-pellet and post 98 °C-supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with heterogeneous nuclear RNA. The hybridization studies showed a different ability of these three DNA preparations in binding nuclear heterogeneous RNA: 16%, 8% and 30% for DNA isolated from 50 °C-pellet, 50–98 °C-pellet and post 98 °C-supernatant, respectively. The results are discussed in terms of chromatin structure and function.

Heterogeneity of mouse unique deoxyribonucleic acid

Kinetic analysis of renaturation has shown that mammalian DNA contains nucleotide sequences which differ in their renaturation rate. The most slowly reassociating component is thought to contain unique sequences, i.e. those present in only one copy in the haploid genome. The rate of renaturation of this DNA fraction in different species correlates with the size of the genome [ 1,2] . The renaturation follows second order kinetics and its course is rather similar to that of E. coli DNA, which does not contain repeated sequences [2] . The thermal stability and the hyperchromism of renatured DNA in this fraction are close to those of the native DNA [ 1,3] . On renaturation these sequences show a strong species specificity [3]. The proportion of DNA defined as unique may vary depending on the conditions of renaturation [4]. The following conditions are accepted as standard for isolation of mammalian unique DNA: fragment size of 200 to 500 base pairs (most often 400-500); incubation at 60” C in 0.12 0.14 M sodium phosphate buffer, pH 6.8 (Nap) and Cot > 200 [5,6]. Under these conditions the unique DNA in the mouse genome amounts to about 60% [ 1,7-91. We found that a considerable heterogeneity can be observed in the unique DNA isolated under standard conditions, when allowing to reassociate at 50°C. The renatured DNA contained at least two fractions in approximately equal proportions differing in their thermal stability. T, of the reassociated duplexes was 12°C below that of the fragmented mouse DNA for the first fraction and 2°C for the second one. The two fractions exhibited different hybridization ability with heterogeneous nuclear RNA and different thermal stability of the RNA-DNA hybrids obtained.

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.

RNA - DNA hybridization on membrane filters with fragmented mammalian DNA.

The possibilities of using fragmented mammalian DNA for hybridization on membrane filters were investigated. The adsorption and release of fragmented DNA were studied as influenced by various factors of the hybridization procedure. It was found that working with preparations sufficiently homogeneous in molecular weight with fragment size 4.8-6.5 S, dissolved in 6 times SSC at neutral pH, the adsorption on filters was almost 100%. After incubation of filters for 18 h in 2 times SSC at 65 degrees C about 50% of the fragmented DNA and 20% of the high molecular weight DNA were released. The degree of release differed for the different families of repeated DNA sequences. Lowest release was obtained with the highly repeated DNA (20%) and highest with the unique DNA (63%), i.e. the release was inversely proportional to the renaturation rate of DNA. In the course of release of fragmented total DNA the material remaining on the filters became enriched in highly repeated sequences, due to selective release of the slowly reassociating fractions. As a result, the percentage of fragmented DNA which hybridized with heterogeneous nuclear RNA was higher than that of high molecular weight DNA. The thermal stabilities of the hybrids with fragmented and high molecular weight DNA were identical. The conditions are defined which permit application of the membrane filter hybridization technique to fragmented mammalian DNA.