Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.     

Two membrane sites for DNA synthesis in Pneumococcus.

Summary A DNA membrane fraction extracted from pneumococci can be separated into two subfractions with respect to macromolecular composition and DNA synthesis by centrifugation in a 30–60% w/v neutral sucrose gradient. Each fraction can be rebanded in a sucrose gradient or centrifuged to equilibrium in a CsCl density gradient without altering the ability of the fractions to synthesize DNA. The fast sedimenting (heavy) fraction contains 45% of the DNA, and the bulk of the phospholipid, protein, and RNA. The light fraction contains 50% of the DNA, and lower, but significant amounts of phospholipid, RNA, and protein. Both fractions contain a DNA replication complex consisting of a number of enzymes involved in synthesizing DNA or DNA precursors, as well as RNA polymerase activity. However, the specific activity of DNA polymerase in the light fraction is much greater than that in the heavy fraction. In addition, the following results suggest that the former is concerned primarily with replication of the genome while the latter has characteristics of a repair function for the genome. (1) newly synthesized DNA can be detected within 30 s in the light fraction but not until 4 min in the heavy fraction. (2) an RNA-DNA single-stranded hybrid can be demonstrated during initial stages of DNA synthesis in the light, but not heavy fraction. (3) extensive semiconservative DNA replication occurs in the light fraction, whereas little such replication is detected in the heavy fraction. (4) DNA polymerase activity in the light fraction has several of the characteristics of a polymerase identified by others as being concerned with normal DNA replication, such as inhibition by N-ethylmaleimide, and relatively high rates of chain elongation (4.9×10⁴ nucleotides/min). In contrast, DNA polymerase activity in the heavy fraction has characteristic properties associated with DNA polymerase I, a possible repair enzyme. These include higher activity for a d(A-T)n template than that detected in the light fraction, no effect of N-ethylmaleimide, and relatively low rates of chain elongation (9×10³ nucleotides/min).     

Fractionation of DNA on benzoylated DEAE-cellulose

Escherichia coli DNA separates into two fractions upon chromatography on columns of benzoylated-DEAE-cellulose (BD-cellulose). One fraction binds more strongly and only elutes in 1m NaCl-1.8% caffeine solution, similar to the behavior of denatured DNA on such columns. Mature λ DNA and supercoiled λ DNA elute mainly in the salt fraction. The bacterial DNA fraction which binds strongly to BD-cellulose behaves as fully native DNA upon CsCl equilibrium centrifugation and hydroxyapatite chromatography but is retained on nitrocellulose filters to a greater extent than the salt-elutable fraction. Treatment of DNA preparations with ribonuclease, pronase, and detergents has no effect on the relative proportions of the two fractions. The possible structural basis for the strong binding of DNA to BD-cellulose is discussed.     

DNA Polymerase Activity Associated with the Membrane Fraction of E. coli

Spheroplasts were disrupted with 0.2% Brij 58 and the separation of intact cells, spheroplasts, disrupted spheroplasts, fragmented membrane, and supernatant was performed on a linear 40~55% sucrose gradient. About half an amount of nucleic acid components was distributed in disrupted spheroplast fractions, while only a small amount of protein components was found in these fractions.

DNA polymerase in the fragmented membrane fraction incorporated ³H-TTP more rapidly than that in the supernatant fraction for the first 5 to 6 min, and then the incorporation rate decreased, while DNA polymerase in the supernatant fraction incorporated ³H-TTP linearly up to 20 min when native DNA was used as a primer. The former required native DNA as a primer and showed little activity towards denatured DNA, while the latter incorporated ³H-TTP at a similar rate to both the primer DNA’s.

DNA polymerase of the fragmented membrane fraction synthesized various sizes of DNA from short to a size of primer when native DNA was used as a primer, while when denatured DNA was used, products were only short. DNA polymerase of the supernatant fraction synthesized various sizes of DNA when both native and denatured DNA’s were used as primers.     

The characteristics of the spindle fiber attachments (SFAs) enriched fraction from mouse nuclei

The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunofiuorescence, test of the activity of microtubule organizing center(MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascites cells. The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD. Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.     

DNA-histone interaction in the vicinity of replication points.

Chromatin replication was studied in isolated nuclei from Concanavalin A activated lymphocytes. Digestion with micrococcal nuclease revealed that the resistant fraction of in vitro replicated DNA is associated with nucleosomes. Earlier experiments had shown that the nuclease resistant fraction of nascent DNA is composed of fragments which are shorter than the nuclease resistant fragments of bulk DNA. In this communication we demonstrate that the short fragments of nascent DNA are differently bound to nucleosome like structures compared to bulk DNA. At 0.5 M NaCl a fraction of pulse labeled labeled DNA is released from these structures and appears as free double stranded DNA of about 140 base pair length (5S DNA) while the 185 pair fragments of mature replicated DNA remain attached to nucleosomes under these conditions. The experiments may indicate that the interaction of a fraction of replicating DNA with histones differs from that of bulk DNA.     

Molecular structure of plant genomes

The genome structure of several species of Graminea and Drosophila was investigated by DNA renaturation method. Kinetics of DNA reassociation was studied by direct optical scanning and the data obout Cot curve were analized by an improved computer programm "Finger". Differences between structure DNA animals and plants are shown. Plant genomes have no unique fraction which exists in animal genomes. Slowly reassociating fraction in plants comprises about 20% DNA as compared with more than 60% in animal DNA. An analysis of kinetic complexity indicates that the relative content of the slowly reassociating fraction in the genome both of animal and of plants is much higher than that of the highly repeated DNA fraction.     

Unmethylated domains in vertebrate DNA.

We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA.     

A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

Objective

The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.

Methods

Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.

Results

A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.

Conclusion

A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.     

Effect of hyperhybridization of nucleic acids]

The reasons for the effect of hyperhybridization (HH) of nucleic acids, when the degree of binding of labelled fragments in the heterologous reaction is higher than in the homologous one, are discussed. The object of investigation was DNA of salmon. HH is most demonstrative in hybridization of a DNA fraction which forms thermostable duplexes (Tm greater than or equal to 75 degrees). HH is accounted for by the fact that closely related species and intraspecies forms differ in the percentage of this fraction; therefore if a species with a small content of this fraction in the genome is chosen as a reference species, hybridization of its DNA with that of another species with a high content of the thermostable fraction, exceeds 100% with respect to the homologous fraction). A correction coefficient is suggested allowing comparison between experiments with different reference species. It appears that the genomes of less specialized, as compared with highly specialyzed species, contain more DNA forming thermostable duplexes. It is therefore recommended to use as reference DNA of species, the structure of karyotype and morphologt of which have more in common with the ancestral form.     

Dna comets as markers of cells death]

Unstimulated human peripheral blood lymphocytes gradually underwent death during incubation in vitro. According to morphological criteria, the type of death was identified as apoptosis. After immobilization in agarose, lysis, and electrophoresis, these lymphocytes formed DNA comets, which differed in DNA content, tail length, tail moment, and the fraction of DNA migrating in the comet tail. We classified the comets in 3 groups in accordance with the values of these parameters. There was a good correlation between the fraction of apoptotic cells (morphological data) and the fraction of "apoptotic" DNA comets. The results showed that DNA comets may be adequate markers of cell death (including apoptosis). The use of DNA comets as markers of spontaneous death made it possible to reveal an increased level of apoptosis in vitro lymphocytes from patients with systemic lupus erythematosus.     

"Endless" viral DNA in cells infected with channel catfish virus

The state of intracellular viral DNA in cells infected with channel catfish virus has been studied by the Hirt selective extraction procedure and by restriction endonuclease digestion. The sedimentation properties and restriction patterns of viral DNA in the Hirt supernatant fraction indicate that the majority, if not all, of the DNA is in the form of linear unit-length (Mr approximately equal to 85 x 10(6)) molecules. However, restriction digests of viral DNA in the pellet fraction lacked two fragments corresponding to the molecular ends of unit-length DNA. In addition, there appeared in HpaI digests of pellet DNA a new restriction fragment interpretable as the product of fusion between the ends of unit-length molecules. The size of the new fragment requires that fusion occur in such a way that one copy of the terminally repeated sequences (Mr approximately equal to 12.3 x 10(6)) of the unit-length DNA is lost in the process. In pulse-chase experiments, radioactivity flowed from the pellet fraction to the supernatant fraction, suggesting a precursor-product relationship for these DNA species. The results are easily understood if unit-length virion DNA is generated by excision from concatemeric structures.     

Polyoma virus minichromosomes: associated enzyme activities.

Polyoma minichromosomes were isolated and fractionated on glycerol gradients as described by Gourlie et al. (J. Virol. 38:805-814, 1981). Specific assays for DNa polymerases alpha, beta, and gamma, DNA topoisomerase I, and RNase H were carried out on each fraction. The number of units of activity in each fraction was compared with the number of total polyoma and replicative intermediate DNA molecules in each fraction determined by quantitative electron microscopy (M. R. Krauss and R. M. Benbow, J. Virol. 38:815-825, 1981). DNA polymerase alpha cosedimented with polyoma replicative intermediate DNA molecules. DNA polymerase beta and DNA topoisomerase I activities sedimented with mature polyoma minichromosomes. Although the bulk of RNase H activity sedimented in the minichromosome region, the peak of activity was found one fraction behind the peak of mature minichromosomes. Virtually no DNA polymerase gamma activity cosedimented with polyoma minichromosomes.     

Polyomavirus minichromosomes: associated DNA topoisomerase II and DNA ligase activities.

Polyomavirus minichromosomes were isolated and fractionated as described previously (B. B. Gourlie, M. R. Krauss, A. J. Buckler-White, R. M. Benbow, and V. Pigiet, J. Virol. 38:805-814, 1981). Specific assays for DNA topoisomerase II and DNA ligase activity were carried out on each fraction. The enzymatic activity in each fraction was determined by quantitative electron microscopy and compared with the number of replicative intermediate and total polyomavirus DNA molecules in each fraction. DNA topoisomerase II activity cosedimented with polyomavirus replicative intermediate minichromosomes. DNA ligase activity cosedimented with mature polyomavirus minichromosomes.     

Transformation and Deoxyribonucleic Acid Size: Extent of Degradation on Entry Varies with Size of Donor

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.     

A group of repetitive human DNA families that is characterized by extrachromosomal oligomers and restriction-fragment length polymorphism

We have recently reported a novel human repetitive DNA (Sau3A family) that exists both in the chromosomes and in the extrachromosomal fraction. Several more clones that hybridized with the Sau3A family were isolated from the extrachromosomal fraction of HeLa cells. Use of these clones as probes has revealed that at least four different types of oligomeric forms of DNA are present in the extrachromosomal fraction. The oligomers consist of one, two, five or 12 subunits of basic 170 base-pair unit DNA, or superimposed forms of two of them. Nucleotide sequencing of these clones indicated that the clones have 70 to 90% sequence homology with human alphoid satellite DNA. These DNA sequences are present also in the chromosomes, as tandemly repeated DNA sequences, and exhibit a considerable degree of restriction-fragment length polymorphism. These results, taken together with the previous findings on Sau3A family DNA, suggest that there is a group of recombination-prone repetitive DNA families in human chromosomes.     

Strandedness and Complementarity of DNA from Long-Term RNA-Dependent DNA Polymerase Reactions of Soehner-Dmochowski Murine Sarcoma Virus

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.     

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells. Alternatively, an individual DNA replication origin sequence - a 478-bp long DNA fragment located at about 17-kb downstream of the dihydrofolate reductase gene - was used to probe the same matrix-attached and loop DNA fractions. The results with both the collective and individual DNA replication origins showed that there was random distribution of the origin sequences between DNA attached to the matrix and DNA from the loops.