The reaction of glyoxal with nucleic acid components III. Kinetics of the reaction with monomers

The restriction endonuclease, HindIll, gives rise to three fragments in each of the three mitochondrial DNAs isolated from the established mammalian cell lines LA9 (mouse), HeLa (human), and BSC-1 (African green monkey). The restriction endonuclease, EcoRI, gives rise to three fragments in mitochondrial DNA from HeLa and to two in DNAS from LA9 and BSC-1. The sizes and the orders of the fragments in the respective genomes have been determined with data obtained from the electron microscope. The origin and the direction of replication have been designated in each of the cleavage maps. Polyacrylamide gel electrophoretic analyses demonstrated that additional fragments not detectable in the electron microscope and larger than 50 nucleotide pairs were not present. The restriction endonucleases EcoRI from Escherichia coli(1) and HindIII from Haemophilus infiuenzae (1) recognize a different hexanucleotide sequence in DNA (1). Each produces a small number of specific fragments with mitochondrial DNAs (mtDNAs) isolated from established cell lines of human, African green monkey, and mouse origin. Because these DNAs contain D-loops at the origin of replication (2, 3), it is possible to develop cleavage maps of the fragments with the sites of the origin of replication as reference points. Since replication proceeds unidirectionally (4, 5), fragments obtained from larger replicating forms disclose the direction of replication on the map and refine the site of origin of replication to one fork of the D-loop. The development of six cleavage maps is described in this communication. It is anticipated that these maps will serve as primary maps for subsequent mapping studies. MATERIALS AND METHODSCell Lines. The established lines were HeLa (human), BSC-1 (African green monkey), and LO9 (mouse Electron Micrographical Analysis of DNA Fragments. The DNA was spread with either aqueous or formamide spreading conditions (7). Grids were stained with alcoholic uranyl acetate and shadowed with Pt-Pd, as described (7). They were examined and photographed in a Philips 300 electron microscope at a (film) magnification of 3000 X. Negatives were projected onto a Hewlett-Packard 9864 A Digitizer connected to a 9820 A calculator system and measured.Either full-length mtDNA or OX174 RF DNA molecules were added to the samples after enzymatic restriction as internal size standards. The contour lengths of the mtDNAs were calibrated against that of OX174 Am3 RF DNA in which 10% of the molecules contained a 7% deletion (8

Section: The Restriction Endonucleases Ecori From Escherichia Colisupporting

confidence: 54%

Restriction Endonuclease Cleavage Maps of Animal Mitochondrial DNAs

Brown

Vinograd

1974

“…3B shows the intensity of the fragments transferred and retained on the nitrocellulose paper through the hybridization and washing conditions described. It is evident that fragments of 200-300 nucleotides (fragments [12][13][14][15][16] are retained with about the same efficiency as the larger fragments of 500-2000 nucleotides. The two smallest fragments (72 and 118 nucleotides) appear in reduced intensity in this autoradiograph.…”

Section: Resultsmentioning

confidence: 86%

Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.

Thomas¹

1980

7,333

2,093

A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCI/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (108 cpm/sg). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently. The technique of Southern (1) enables one to transfer electrophoretically separated DNA fragments to nitrocellulose paper for hybridization with specific radioactive DNA or RNA probes. Although RNA does not generally bind to nitrocellulose, it has been transferred and covalently coupled to activated cellulose paper (diazobenzyloxymethyl-paper, DBM-paper) according to the method of Alwine et al. (2,3). In our laboratory, the use of activated paper for coupling RNA has been complicated by two major problems: first, about 500 pg of specific RNA per band is just detectable (after several days exposure) after hybridization using high specific activity probes prepared by nick translation (108 cpm/,ug). Although this sensitivity is similar to that obtained by others with this method (2, 3), we estimate that this is 1-10% of that for detecting specific DNA sequences on nitrocellulose by using similar probes. The increased sensitivity of the nitrocellulose paper is probably a reflection of the fact that nitrocellulose has a higher binding capacity for DNA (about 80 ,Ug/cm2) compared to the capacity of most preparations of activated paper for binding ,gg/cm2). Second, we have found that preparation and activation of DBM-paper is expensive, time consuming, and, most importantly, often variable.Poly(A)+ RNA is retained on Millipore filters in 0.5 M KCI (4, 5). We therefore decided to investigate whether RNA in general could be bound to nitrocellulose paper and retained during hybridization and stringent washing. Here I describe a rapid and simple method for blot hybridization of denatured The publication costs of this article were defrayed in part by page charge pa...

“…At pH 7, the glyoxal-guanosine adduct is stable for at least 20 hr at 20° (9). This means that the gel or running buffers do not have to contain denaturing agents, thus making it possible to analyze both native and fully denatured molecules on the same slab gel.…”

Section: Discussionmentioning

confidence: 98%

Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange.

McMaster¹,

Carmichael²

1977

2,070

664

We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 500. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double-and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel. The electrophoretic mobility of nucleic acids in polyacrylamide or agarose gels depends on both molecular weight and conformation (1, 2). Removing secondary and tertiary structure should make the electrophoretic mobility a simple function of molecular weight. Gels containing the denaturing agents formaldehyde (3), formamide (4,5), methylmercuric hydroxide (6), and urea (7) have all been used for molecular weight determinations. Here we present a simple and convenient method that we feel offers a number of advantages over those previously described. This method employs denaturation of nucleic acids and reaction with glyoxal, followed by electrophoresis in a slab gel.