Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
TL;DR: In this article, the rat pancreas RNA was used as a source for the purification of alpha-amylase messenger ribonucleic acid (RBA) using 2-mercaptoethanol.
Abstract: Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
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TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
65,881 citations
TL;DR: The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.
Abstract: The mechanisms that balance food intake and energy expenditure determine who will be obese and who will be lean. One of the molecules that regulates energy balance in the mouse is the obese (ob) gene. Mutation of ob results in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.
12,394 citations
TL;DR: A role for TNF-alpha in obesity and particularly in the insulin resistance and diabetes that often accompany obesity is indicated.
Abstract: Tumor necrosis factor-alpha (TNF-alpha) has been shown to have certain catabolic effects on fat cells and whole animals. An induction of TNF-alpha messenger RNA expression was observed in adipose tissue from four different rodent models of obesity and diabetes. TNF-alpha protein was also elevated locally and systemically. Neutralization of TNF-alpha in obese fa/fa rats caused a significant increase in the peripheral uptake of glucose in response to insulin. These results indicate a role for TNF-alpha in obesity and particularly in the insulin resistance and diabetes that often accompany obesity.
7,347 citations
TL;DR: A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis agent and screened with serum from a patient diagnosed with NANBH, showing consistent with the agent being similar to the togaviridae or flaviviridae.
Abstract: A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.
6,814 citations
TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients
6,731 citations
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TL;DR: The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature.
Abstract: Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).
13,649 citations
TL;DR: A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described and should prove generally useful as an initial step in the isolation of specific mRNAs.
Abstract: A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.
5,833 citations
TL;DR: A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system, and no residual nuclease activity could be detected, and the tRNA is functionally unimpaired.
Abstract: A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N′-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.
3,591 citations
2,054 citations
TL;DR: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA that contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin II prepeptide, and the untranslated 3' terminal region of the mRNA.
Abstract: Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.
1,135 citations