The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.

Rapid transfer of DNA from agarose gels to nylon membranes.

https://www.cell.com/cell/pdf/0092-8674(90)90395-U.pdf

Antitumor promotion and antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone

Cloning and sequence analysis of cDNA for the Electrophorus electricus electroplax sodium channel indicate that this protein, consisting of 1,820 amino acid residues, exhibits four repeated homology units, which are presumably oriented in a pseudosymmetric fashion across the membrane. Each homology unit contains a unique segment with clustered positively charged residues, which may be involved in the gating structure, possibly in conjunction with negatively charged residues clustered elsewhere.

Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence.

Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1

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 50 degrees. 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.

https://www.pnas.org/content/pnas/74/11/4835.full.pdf

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

Publisher Summary This chapter discusses the analysis of nucleic acids in gels using glyoxal and acridine orange. To determine the molecular weights of nucleic acids using gel electrophoresis techniques, the molecules must have equivalent conformations. This is achieved by removing native secondary and tertiary structure with a variety of chemical denaturants, thus reducing the electrophoretic mobility to a simple function of polynucleotide molecular weight, gel composition, and voltage gradient applied. The chapter describes the method for denaturing RNA or DNA molecules with glyoxal, followed by electrophoresis through either polyacrylamide- or agarose-containing slab gels in a low ionic strength buffer. Using this method reliable molecular weight estimates for RNA and DNA molecules of varying sizes and G + C contents were obtained; glyoxalated DNA and RNA molecules were shown to lie on the same log molecular weight versus mobility curve, or on very similar curves. The chapter describes the use of the metachromatic stain, acridine orange, for the visualization of nucleic acids in gels. When intercalated between the stacked bases of double-helical nucleic acids, acridine orange manifests a green fluorescence. Its metachromasy visually confirms glyoxal denaturation and makes it a powerful reagent for rapid determination of gross nucleic acid structure after gel electrophoresis.

Gary K. McMaster

Papers

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

[47] The analysis of nucleic acids in gels using glyoxal and acridine orange