The use of buoyant density profiles in determining the length and spacing of repeated sequences in DNA

Hearst, John E.; M, Botchan; Kram, Raphaël; Beals, E.

doi:10.1016/0005-2787(73)90290-6

Cited by 6 publications

(2 citation statements)

References 9 publications

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“…This class can be distinguished from the other two in that there will be a large fraction of the total fragment which will anneal with itself or with total DNA only at a high Cot. Evidence for such a model of interspersed unique and reiterated DNA has been recently reported, and the calculations on the relative sizes and reiteration frequencies of these regions (5)(6)(7)(8) are compatible with some of the fragments reported here (Table 1).…”

Section: Discussionsupporting

confidence: 68%

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Generation of Specific Repeated Fragments of Eukaryote DNA

Mowbray¹,

Landy²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Calf-thymus DNA, hydrolyzed with a sitespecific endonuclease from Haemophilus influenzae Rd, yields 12 discrete bands on polyacrylamide-agarose gels. These range in size from 7.5 X 104 to 2 X 106 daltons, and they represent about 5% of the total DNA with individual fragments comprising 0.1-1.5%. We have studied repetitive sequences in calf-thymus DNA using a site-specific endonuclease (restriction enzyme) prepared from Haemophilus influenzae Rd (9). This enzyme fraction [abbreviated Endo R-Hind (10)], which consists of two site-specific nucleases (11), makes double strand scissions in the DNA at specific recognition sites (restriction sites).Assuming that there are at least two nuclease recognition sites in a repeated nucleotide sequence, the number of copies of the specific DNA fragments produced from that sequence will be a function of the number of times it appears in the genome. When enzymatically hydrolyzed DNA is fractionated on polyacrylamide-agarose gels, some fragments derived from repeated sequences can be identified as discrete bands, whereas fragments from unique sequences are present as a continuous distribution of various size DNA fragments (Fig. 2). The size distribution of the fragments is, of course, determined by the spacing of the restriction sites within the DNA. METHODSIsolation of DNA. Calf-thymus tissue was homogenized and the nuclei were isolated using a modification of the procedures of Allfrey et al. (12). A Teflon-glass homogenizer was used to disrupt the tissues and break the cells. Nuclei were purified by centrifugation through 2.4 M sucrose in 3 mM CaCl2, 10 mM Tris * HCl (pH 7.4) and were subsequently disrupted by incubation (12-15 hr) in sodium dodecyl sulphate (0.5%), Pronase (100,gg/ml), 50 mM KCl, 10 mM Tris HCl (pH 8.0). DNA was extracted with an equal volume of chloroformisoamyl alcohol (24:1) at least three times, dialyzed, incubated with T, and pancreatic RNAase (60 units/ml each), treated with pronase, and extracted with phenol.Enzymatic Hydrolysis of DNA. Calf DNA was hydrolyzed with Endo R-Hind as previously described (11). Conditions, chosen such that the reaction was complete, were about 0.5 units of enzyme (9) per ml added at 11/2 hourly intervals for a total of three additions.Polyacrylamide Gel Electrophoresis. The DNA fragments produced with Endo R * Hind are routinely separated on composite polyacrylamide-agarose slab gels (3% acrylamide, 0.5% agarose) and stained as previously described (11). Electrophoresis was usually for 41/2 hr at 200 V in a slab gel electrophoresis cell cooled to 2-4°. The amount and position of DNA was estimated by optically scanning a gel at 590 nm followed by integration of the area in a graphic read-out of the scan.

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Section: Discussionsupporting

confidence: 68%

“…Recent studies indicate that some of the reiterated DNAs are interspersed between unique sequences. In the case of Drosophila, Necturus, and mouse DNAs, these interspersed, repetitive sequences appear to be distinct from the density satellites (5)(6)(7)(8).…”

mentioning

confidence: 99%

Generation of Specific Repeated Fragments of Eukaryote DNA

Mowbray¹,

Landy²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Mathematical method for determining the distribution of substituted DNA zone lengths with reference to equilibrium density profiles

Delcroix¹,

Cantraine²,

Hardt³

1978

Biopolymers

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SynopsisA method of calculation to determine the profile in density-gradient sedimentation of bromodeoxyuridine-substituted DNA lengths prior to sonication is developed. The method may he summarized as follows: (1) A description of a formulation of random cleavage of a substituted zone i in fragments of length m; (2) generalization and application of this formulation to all substituted zone lengths simultaneously present in a given DNA extract, such generalization being performed by means of a matrix calculation; (3) adjustment of this formulation for the limits of the experimental method, i.e., number of gradient fractions which can he recovered, and for the evolution of the DNA fragment lengths which drop in the course of successive sonications; (4) simulation of the radioactive profiles. By this method it is possible to obtain certain indications concerning the types of initial (i.e., presonication) distributions that are compatible with observed profiles and to test the validity of assumptions relating to the initial distribution. This method has been applied in analyzing DNA synthesis in lymphocytes stimulated by Concanavalin and allowed us to determine the significance of the discontinuities appearing in the elongation with a suboptimal stimulating dose. The proposed method could also he applied to the analysis of all random populations of mixed sequences. INTRODUCTIONA method commonly used to determine the existence and the length of DNA segments newly synthesized either during m i t~s i s l -~ or by repair4 is based on replacing thymidine in the culture medium by bromodeoxyuridine (BrdUrd). The DNA is subsequently extracted and analyzed by densitygradient centrifugation. The results are difficult to analyze because DNA extraction introduces random breaks both in the substituted and nonsubstituted segments. The end result is a population of molecules, generally of homogeneous size but with varying BrdUrd-substituted zone fractions. Thus, it is difficult to determine the initial lengths of the substituted zones based on the characteristics of the population of observed molecules.1,2In general, in order to check the assumed initial distribution of substituted lengths, different sedimentation profiles of the same material are

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