Detection and resolution of closely related satellite DNA sequences by molecular cloning.

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm³ satellite DNA of Drosophila melanogaster into two distinct components: poly$\text{d}\text{A-A-G-A-G}\text{T-T-C-T-C}$ and poly$\text{d}\text{A-A-G-A-G-A-G}\text{T-T-C-T-C-T-C}$. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.     

Restriction endonuclease analysis and nucleotide composition of satellite III DNA from Drosophila virilis

Satellite III DNA from $\text{Drosophila virilis}$ is composed of a tandemly repeated heptanucleotide containing the sequence which is normally cleaved by EcoRI¹ restriction endonuclease activity. However, we observed only a very limited amount of cleavage of satellite III DNA by this activity. Although methyldeoxyadenosine is known to block EcoRI¹ activity, no modified nucleotides were detected in satellite III DNA subjected to nucleotide composition analysis. Since the proportion of each nucleotide present was consistent with the heptanucleotide sequence, we speculate that the resistance of satellite III DNA to EcoRI¹ cleavage may result from the highly repetitive nature of this DNA.     

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.     

Evidence for the attachment of RNA to pulse-labeled DNA in the slime mold, Physarum polycephalum

When $\text{Physarum}\text{polycephalum}$ is pulse-labeled for up to 20 minutes with ³H-thymidine and the shortest labeled DNA strands are partially purified by sedimentation through a neutral aqueous sucrose gradient and then through a formamide-sucrose gradient, these short strands band in Cs₂SO₄ isopycnic density gradients at a density greater than that of bulk single-stranded DNA. Their density is brought partially or nearly completely back to that of single-stranded DNA by hydrolysis with pancreatic RNase A or alkali, respectively. Therefore the dense material attached to the short pulse-labeled DNA strands consists at least partially of RNA.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm³, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm³ appear to be heterogeneous.     

Changes in structural integrity of heart DNA from aging mice

The state of the structural integrity of the DNA from mouse myocardial cells has been investigated by utilizing both CsCl density gradient sedimentation and digestion by S₁ endonuclease from $\text{Aspergillus}$$\text{orzae}$. The DNA from myocardial cells of young mice sedimented in a narrow peak at the expected density of 1.701 g/cm³, while the DNA from the heart cells of senescent mice became broadly distributed in CsCl gradients, banding even more multimodally in alkaline sucrose gradients. This mode of sedimentation indicates that old mouse DNA becomes partially fragmented. When the native DNA of myocardial cells from 6, 20 and 30 month old mice was treated with single-strand specific S₁ endonuclease, it was the DNA from the senescent mice that showed a progressive increase in sensitivity to digestion by the enzyme. The results indicate that the heart DNA of aging mice develops single-stranded gaps in addition to a breakdown into differently sized fragments.     

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

The DNA containing the genes for rRNA (commonly called rDNA) of $\text{Tetrahymena}$ sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with Ø29 phage DNA (M.W. = 11 × 10⁶). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 10⁶.     

Replication of repeated DNA in human cells

The replication pattern of the repeated sequence families of human DNA has been studied by means of DNA reassociation curves. Early- and late-replicating DNA fractions were obtained from synchronized cultures of KB cells by labeling cells with bromodeoxyuridine (BUdR) early or late in the DNA synthesis period and isolating the BUdR-containing DNA by CsCl density-gradient centrifugation. Highly repeated and moderately repeated sequence classes labeled with ${}^{14}\text{C-deoxycytidine}$ either early or late in the DNA synthesis period were also prepared. The effect of the isolated early- or late-replicating BUdR-DNA on the rate of reassociation of the ${}^{14}\text{C-labeled}$ repeated sequences was then tested. Increasing concentrations of early- or late-replicating BUdR-DNA were added to a constant amount of either ${}^{14}\text{C-labeled}$ early- or late-replicating repeated sequences, and the fraction of label in double-stranded DNA was determined. Analysis of the DNA reassociation curves so obtained indicates that some repeated sequence families are replicated throughout the DNA synthesis period whereas others are replicated primarily in the second half. This is true for both the highly-repeated and moderately-repeated sequence classes.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: Localization in polytene and metaphase chromosomes

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664g/cm³, while D. n. albomicana has at least three satellites with densities of 1.674g/cm³, 1.665g/cm³ and 1.661 g/cm³. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

Covalent binding of ethidium azide analogs to Salmonella DNA in vivo: competition by ethidium bromide.

The photoreactive analogs of ethidium bromide (ethidium mono- and diazide) have been developed as drug probes to determine the actual molecular details of ethidium bromide interactions with DNA. In an effort to demonstrate that the analogs in fact mimic the parent ethidium, competition experiments were designed using ³H thymidine-labeled DNA in intact $\text{Salmonella}$ TA1538, which is reverted by the azide analogs. ¹⁴C-labeled ethidium azide analogs were used in combination with the non-labeled ethidium bromide. The results presented here demonstrate that the parent ethidium competes with the azide analogs as a DNA intercalating drug using CsCl density gradient ultracentrifugation.     

Localization of repeated DNA sequences in CsCl gradients by hybridization with complementary RNA

Single-stranded ends of mouse satellite DNA sequences exposed during the extraction of nuclear DNA, or by mechanical shear, can hybridize with highly labelled RNA, complementary to satellite DNA. In neutral CsCl gradients, the hybrid sediments as a sharp peak at the density of mouse satellite DNA (1.691 g/cm³). The possibility of exploiting this observation in locating and isolating cryptic repetitive DNAs from nuclear DNA or the DNA of heterochromatin is discussed.     

Analysis of DNAs from two species of the virilis group of Drosophila and implications for satellite DNA evolution

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.