Fractionation of eukaryotic DNA in a pulsating electrical field. I. Detection and properties of discrete DNA fragments]

The fractionation of eukaryotic DNA by field inversion gel electrophoresis results in the appearance of discrete DNA-fragments. The set of these fragments is similar to that of different eukaryotic representatives and consists of various chromosomal DNAs, unified by size. The physical properties of DNA-fragments suggest that they can form multimeric structures due to the presence of sticky ends flanking discrete fragments. We suppose that the set of discrete DNA-fragments results in a specific cleavage of intact nuclear DNA and can reflect different levels of chromatin structural organization.     

Effect of the conditions of isolation and incubation of the nuclei on digestion of DNA chromatin by calcium- and magnesium-dependent endonuclease. Change in the spectrum of chromosomal proteins and possible role of DNA-protein interactions]

Digesting of chromosomal DNA of interphase rat liver nuclei by Ca, Mg-dependent endonuclease in situ in the presence of chelating agents results in the appearance of the soluble DNP--up to 30% of the total DNA. In addition, 50% of the chromatin is solubilised after mild ultrasonication. In the absence of the chelating agents the degree of fragmentation is considerably increased. The process is accompanied by a loss of some histone and nonhistone chromosomal proteins; the nonhistone proteins are lost selectively. The preliminary removal of the nuclear membrane and significant part of the proteins by tritone X-100 promotes the chromatin degradation and the appearance of low molecular weight fragments. The DNA-fragments of solubilised chromatin are similar to the DNA-fragments of residual chromatin, but in the presence of the chelating agents the latter does not contain monomeric fragments.     

Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA.

A new method for isolating DNA from agarose gels is described. The method involves the simultaneous transfer of all DNA-fragments from an agarose slab gel onto DEAE-cellulose paper and the elution of the individual fragments from the paper with 1 M NaCl. DNA isolated from agarose gels in this way is susceptible to cleavage with several restriction endonucleases, and can be labeled in vitro with E coli DNA-polymerase I, T4 DNA-polymerase and T4 polynucleotide kinase. We have used the method to construct restriction endonuclease maps of adenovirus type 16 DNA.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication.

DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.     

Classification of Normal and Male-Sterile Cytoplasms in Maize. II. Electrophoretic Analysis of DNA Species in Mitochondria

Mitochondrial DNA preparations were made from 31 maize lines carrying different sources of cytoplasm in the same nuclear genetic background. The DNAs were analyzed by agarose gel electrophoresis. A number of discrete low molecular weight bands were present in all lines. However, only four different DNA banding patterns were observed. These were correlated with the N, T, S and C cytoplasms defined by nuclear fertility restorer genes. Of the 31 cytoplasmic sources examined, six possessed DNA species characteristic of N cytoplasms, four possessed DNA species characteristic of T cytoplasm, 19 possessed DNA species characteristic of S cytoplasm and two possessed DNA species characteristic of C cytoplasm. This classification is in complete agreement with that based on mitochondrial translation products reported in the accompanying paper. No within-group heterogeneity was observed in the DNA banding patterns, indicating a lack of cytoplasmic variation within the four cytoplasmic groups. Attributes of the various methods available for classifying maize cytoplasms are compared and discussed.     

Clustering of the DNA sequences complementary to repetitive nuclear RNA of HeLa cells.

We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.     

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Proliferating cell nuclear antigen is required for DNA excision repair.

Fractionation of extracts from human cell lines allows nucleotide excision repair of damaged DNA to be resolved into discrete incision and polymerization stages. Generation of incised intermediates depends on the XP-A protein, a polypeptide that recognizes sites of damaged DNA, and on the human single-stranded DNA-binding protein HSSB. The proliferating cell nuclear antigen (PCNA) is required for the DNA synthesis that converts the nicked intermediates to completed repair events. This need for PCNA implies that repair synthesis is carried out by DNA polymerase delta or epsilon. The ability to visualize repair intermediates in the absence of PCNA facilitates dissection of the multiprotein reaction that leads to incision of damaged DNA in a major pathway of cellular defense against mutagens.     

Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.     

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.     

Construction and assembly of branched Y-shaped DNA: "click" chemistry performed on dendronized 8-aza-7-deazaguanine oligonucleotides

Branched DNA was synthesized from tripropargylated oligonucleotides by the Huisgen-Meldal-Sharpless cycloaddition using "stepwise and double click" chemistry. Dendronized oligonucleotides decorated with 7-tripropargylamine side chains carrying two terminal triple bonds were further functionalized with bis-azides to give derivatives with two terminal azido groups. Then, the branched side chains with two azido groups or two triple bonds were combined with DNA-fragments providing the corresponding clickable function. Both concepts afforded branched (Y-shaped) three-armed DNA. Annealing of branched DNA with complementary oligonucleotides yielded supramolecular assemblies. The concept of "stepwise and double click" chemistry combined with selective hybridization represents a flexible tool to generate DNA nanostructures useful for various purposes in DNA diagnostics, delivery, and material science applications.     

The DNA of Volvox carteri: a biophysical and biosynthetic characterization.

Various biophysical and biosynthetic characteristics of deoxyribonucleic acid (DNA) from Volvox carteri are examined. The DNA from three strains (HK-10,NB-7 and KA-1) is compared, and all strains are shown to contain at least two distinct DNA species which band at densities of 1.714-1.715 and 1.704-1.705 g/cm3 in neutral CsCl and correspond to nuclear and "cytoplasmic" DNA, respectively. Base compositions calculated from these densities, 55-56% G+C for nuclear DNA, and 45-46% G+C for cytoplasmic DNA, are in close agreement with % G+C values estimated from thermal denaturation data. DNA from strain KA-1 has a third component with a buoyant density of 1.693 g/cm3. DNA synthesis is analysed using radioactively labelled heterogeneously grown strains of Volvox carteri and profiles obtained following preparative CsCl density gradient centrifugation are presented. In addition, dissimilarities in patterns of DNA synthesis at various periods in the asexual life cycle are reported for synchronous cultures of strain HK-10. These differences in temporal patterns of DNA synthesis clearly indicate that while nuclear DNA is make to some degree throughout the life cycle, cytoplasmic DNA synthesis appears to occur only at discrete intervals.     

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.     

Two-dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum. Developmental regulation of the ribosomal gene number

The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high-resolution gel analysis. Three types of repeated DNA fragments in the size range of (0.2-2) X 10(3) base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above-average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T). On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)-rich repeat fractions, whereas several of the (G + C)-rich fractions revealed fewer copies in the DNA prepared from starved cells. As shown by hybridization under saturating conditions, the reduction of several (G + C)-rich repeated sequences in the restricted nuclear DNA in sporulation-competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.     

Identification of three populations ofOphiostoma ulmi (aggressive subgroup) by mitochondrial DNA restriction-site mapping and nuclear DNA fingerprinting

Three genetically distinct populations of the Dutch elm pathogenOphistoma ulmi within the aggressive subgroup were defined by the hybridization of a human minisatellite DNA sequence (HVR 33.6) to polymorphic dispersed DNA sequences within theO. ulmi nuclear genomes. For the 10 isolates examined there was a close correlation between nuclear DNA fingerprints and mitochondrial (mt) DNA restriction patterns. A restriction-site map was constructed for the mitochondrial genomes for each of these populations. The three mt DNA maps corresponded to genome sizes of 49.1 (Type I), 49.9 (Type II), and 53.9 (Type III) kilobase pairs (kbp) of DNA. The Type I and Type II mt genomes differed from the Type III mt genome by discrete length mutations of 4.8 and 4.0 kbp, respectively. It is unknown whether these length mutations resulted from insertions into or deletions from a progenitor mitochondrial genome. There was no correlation between the mitochondrial or nuclear genotypes and the geographical source of the isolates.     

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.