Fractionation of eukaryotic DNA in a pulsed electrical field. II. Discrete DNA fragments and level of structural organization of chromatin]

The data presented confirm the possibility of enzymatic formation of discrete DNA-fragments appearing during fractionation of nuclear DNA by FIGE. Teniposide-dependent pattern of DNA-fragments as well as occurrence of protein-linked DNA breaks suggest that discrete cleavage of intact nuclear DNA is modulated by DNA topoisomerase II. The possible relationship between discrete DNA-fragments and the higher order chromatin folding are discussed.     

Detection of left-helical segments in eukaryotic DNA]

The method of DNA binding to nitrocellulose filters was applied to DNA isolated from mouse liver and Ehrlich ascite carcinoma (EAC), calf thymus, and lymphocytes from patients with chronic lymphoid leukemia. In those and phage PM2 DNA the increase in the DNA binding to the filters with a rise in NaCl concentration from 0.5 up to 4.5 M was sigmoidal being suggestive of a conformational transition. No such activity was found in the case of phage lambda or single-stranded DNA. The binding decreased dramatically after mild cleavage of DNA with DNAase I or treatment with phospholipase C or Eco RI and Hin PI restrictases. Incubation of DNA with ethidium bromide led to decrease in the amount of bound DNA. This effect was enhanced with a rise in the dye concentration. The isotherms of ethidium bromide binding to eukaryotic DNA obtained in Scatchard plots by optic titration had a component with a positive slope at low values of r. Bivalent ions (Mg2+, Zn2+) shifting the equilibrium towards the Z-form increased the proportion of macromolecules retained on the filters at NaCl concentrations of 1-3 M. Local changes in the helix conformation were studied with the help of chemical probes: diethylpyrocarbonate (guanine Z-DNA) and osmium-pyridine reagent (pyrimidines of boundary B-Z sites). These probes incorporation into samples of liver DNA, EAC, and lymphocytes resulted in chemical modification of all these samples. Modification of DNA by osmium-pyridine reagent led to inhibition of subsequent restriction by Eco RI restrictase. The data obtained are suggestive of the presence of Z-regions in the B-helix of eukaryotic DNA. A topological model of Z-site stabilization in small superhelical loops of DNA fixed by protein or lipoprotein molecules is proposed.     

Cloning chloroplast DNA in escherichia coli. I. Construction and selection of recombinant plasmids containing fragments of pea chloroplast DNA]

Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained.     

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.     

Detection in situ of foreign DNA in eukaryotic cells

A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.     

Fractionation of xyloglucan fragments and their interaction with cellulose.

Tamarind seed xyloglucan was partially degraded with a purified endoglucanase (endoV) from Trichoderma viride. Analysis by high-performance anion-exchange chromatography showed that this digest was composed of fragments consisting of 1 to 10 repeating oligosaccharide units ([xg]1-[xg]10). To study the adsorption of xyloglucan fragments to cellulose in detail, this digest was fractionated on BioGel P-6. Fragments were separated satisfactorily up to 5 repeating oligosaccharide units ([xg]5). The galactose substitution of the fragments increased with increasing molecular weight. The BioGel P-6 pools, as well as polymeric xyloglucan ([xg] infinity), were tested for their ability to interact with Avicel crystalline cellulose. Quantitative binding to cellulose occurred for sequences consisting of (at least) 4 repeating units. The adsorption of [xg]4 to Avicel was very high relative to that of [xg] infinity. The dimensions of these fragments were such that they could also penetrate the smaller pores of cellulose. Apparently, the effective surface area for the polymers is much smaller. Adsorption isotherms of [xg] infinity and [xg]4 showed a pattern that is typical for polydisperse systems. However, the mechanisms underlying these patterns were different. At high xyloglucan concentrations, this polydispersity resulted in preferential adsorption of the larger molecules in the case of [xg] infinity and a more extensive colonization of the smaller pores of cellulose in the case of [xg]4. The pH influenced the interaction between xyloglucan (fragments) and cellulose to only a small extent.     

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme.nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.     

Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins.

A DNA topoisomerase activity is found to be associated with the nucleosomes released by the Staphylococcal nuclease digestion of HeLa nuclei. Such an association is found to be salt dependent. A number of criteria have established that this DNA topoisomerase activity is due to HeLa topo I (Liu, L. F. and Miller, K. G. (1980) Proc. Natl. Acad. Sci. USA 78, 3489-3491). A similar association has been demonstrated from the in vitro studies using purified mononucleosomes and eukaryotic DNA topoisomerase I. Nonhistone HMG proteins and histone H1 are found to stimulate topoisomerase activity in vitro and form tight complexes with eukaryotic DNA topoisomerase I. The intimate interactions of topoisomerase I with chromosomal proteins and nucleosomes may be an essential feature of the topoisomerase function in vivo.     

Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA

A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.     

FAB immunoglobulin fragments. I. The comparative characteristics of the serological and virus-neutralizing properties of a gamma globulin against tick-borne encephalitis and of the FAB fragments isolated from it]

A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a marked virus-neutralizing activity. The mean value of a logarithm of the neutralization index was 2.65 +/- 0.2 for Fab-fragments and 3.74 +/- 0.38 for gamma-globulin (P less than 0.01).     

The effect of discrete charges on the electrical properties of membranes. II

In this paper we calculate the hydrogen ion titration curves and associated surface potential for a lattice of discrete sites by numerically solving a titration equation constructed by expanding the partition function for the lattice in powers of the density of charged groups. The titration equations were calculated for several different lattice spacings and ionic strengths of the bathing solution. The results of applying this theory to several simple models of the membrane-solution interface is compared to the corresponding results obtained via the uniform-charge model.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. I. Noncanonical structures

We report results of the first systematic study of conformational polymorphism of G-rich DNA fragments of Alu-repeats. Alu retrotransposons are primate-specific short interspersed elements. Using the Alu sequence of the prooncogen bcl2 intron and the consensus AluS_x sequence as representative examples, we have determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites—PQSAlu), and demonstrated by bioinformatics methods that these sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 bp). These sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element of the human bcl2 gene (304 bp) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alu_bcl2 dsDNA and their absence in the case of Alu-PQS have been shown using DMS-footprinting and atomic force microscopy (AFM). Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions of the reporter gene have been prepared, and their regulatory effects have been compared during transfection of НЕК293 and HeLa cells. We suggest that the dynamic study of the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements responsible for the development of many oncological and neurodegenerative diseases.     

Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.

Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (H.A.-Red) with high G/C specificity is suggested which may be as effective in detecting other sequence changes.     

R.SspD5I is a neoschizomer of HphI producing blunt end DNA fragments

The strain Staphylococcus species D5 produces a restriction enzyme. It is the neoschizomer of HphI endonuclease, which cleaves DNA at a distance of eight nucleotides from the recognition sequences producing blunt end DNA fragments: 5'-GGTGA8N /-3' and 3'-CCACT8N /-5'.     

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.     

Recombination of a eukaryotic DNA in bacteria

Single, 824 bp repeating units of Xenopus laevis oocyte-type 5S DNA were inserted into the recombination vectors, λrva and λrvb. When the inserts had the same orientation with respect to the λ chromosomes, Spi^-imm434 recombinants were recovered by selection on a P2, λ double lysogenic host. Because of the structure of the vectors, the crossover point in each recombinant must lie completely within the 5S DNA insert. The physical characteristics of these recombinants were determined by examination of restriction enzyme digests. By use of RecA mutant hosts and the Red^- vector, λrvc, recombination frequencies were measured separately for the bacterial and phage systems.Some of the recombination events resulted in 5S DNA inserts of altered length due to unequal crossovers within repeated sequences in the 5S DNA spacer. The occurrence of just such events in frog 5S DNA had been predicted, based on the structure of 5S DNA and evolutionary considerations.