scholarly journals Characterization and use of the potent ribonuclease inhibitor aurintricarboxylic acid for the isolation of RNA from animal tissues

1989 ◽  
Vol 263 (1) ◽  
pp. 73-80 ◽  
Author(s):  
A F Skidmore ◽  
T J C Beebee
Keyword(s):  
Nucleic Acids ◽  
Northern Blotting ◽  
Rat Tissues ◽  
Agarose Gel ◽  
Animal Tissues ◽  
Ribonuclease Inhibitor ◽  
Subsequent Removal ◽  
Aurintricarboxylic Acid ◽  
High Yields

Inclusion of aurintricarboxylic acid (ATA) in extraction buffers for the isolation of RNA from animal tissues resulted in high yields (0.5-2.0 mg/g of tissue) of undegraded material as judged by agarose-gel-electrophoretic analyses and Northern-blotting experiments. However, ATA bound to nucleic acids, forming stable complexes, and so we have established methods for spectrophotometric quantification of RNA in these coloured complexes, and for easy removal of sufficient ATA to leave RNA in a consistently hybridizable condition at the end of a purification. The use and subsequent removal of ATA was straightforward and gave satisfactory results for all rat tissues tested.

scholarly journals Synthesis and Antiproliferative Evaluation of 2-Deoxy-N-glycosylbenzotriazoles/imidazoles

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3742
Author(s):  
Caleigh S. Garton ◽  
Noelle K. DeRose ◽  
Dylan Dominguez ◽  
Maria L. Turbi-Henderson ◽  
Ashley L. Lehr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Addition Reaction ◽  
Protecting Group ◽  
Human Breast ◽  
Lung Cancer Cell ◽  
Subsequent Removal ◽  
Anticancer Properties ◽  
Tributyltin Hydride ◽  
High Yields

A series of 2-deoxy-2-iodo-α-d-mannopyranosylbenzotriazoles was synthesized using the benzyl, 4,6-benzylidene and acetyl protected D-glucal in the presence of N-iodosuccinimide (NIS). Subsequent removal of the iodine at the C-2 position using tributyltin hydride under free radical conditions afforded the 2-deoxy-α-d-glucopyranosylbenzotriazoles in moderate to high yields. This method was extended to the preparation of substituted 2-deoxy-β-d-glucopyranosylimidazoles as well. The stereoselectivity of the addition reaction and the effect of the protecting group and temperature on anomer distribution of the benzotriazole series were also investigated. The anticancer properties of the newly synthesized compounds were evaluated in a series of viability studies using HeLa (human cervical adenocarcinoma), human breast and lung cancer cell lines. The N-[3,4,6-tri-O-benzyl-2-deoxy-α-d-glucopyranosyl]-1H-benzotriazole and the N-[3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranosyl]-2H-benzotriazole were found to be the most potent cancer cell inhibitors at 20 µM concentrations across all four cell lines.

Multiple calmodulin mRNA species are derived from two distinct genes

1987 ◽  
Vol 7 (5) ◽  
pp. 1873-1880
Author(s):  
H Nojima ◽  
K Kishi ◽  
H Sokabe
Keyword(s):  
Dna Sequences ◽  
Northern Blotting ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Rat Tissues ◽  
Coding Region ◽  
Genomic Libraries ◽  
Mrna Species ◽  
Nuclease Mapping ◽  
Rat Genome

We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).

Detection of HIV-1 Nucleic Acids by Northern Blotting

HIV Protocols ◽  
2003 ◽  
pp. 71-82
Author(s):  
Richard A. McDonald ◽  
Christopher A. D. Smith
Keyword(s):  
Nucleic Acids ◽  
Northern Blotting ◽  
Hiv 1

Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis

2005 ◽  
Vol 43 (8) ◽  
Author(s):  
Qing Huang ◽  
Wei-Ling Fu
Keyword(s):  
Comparative Analysis ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Ethidium Bromide ◽  
Gel Electrophoresis ◽  
Fluorescent Dyes ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Dna Staining

AbstractEthidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualization of nucleic acids. Safer nucleic acid stains, such as SYBR

Rat Muscle Fructose-1,6-Bisphosphatase: Cloning of the cDNA, Expression of the Recombinant Enzyme, and Expression Analysis in Different Tissues

1999 ◽  
Vol 380 (9) ◽  
pp. 1079-1085 ◽  
Author(s):  
Samiya Al-Robaiy ◽  
Klaus Eschrich
Keyword(s):  
Northern Blotting ◽  
Recombinant Enzyme ◽  
Rat Tissues ◽  
Mrna Levels ◽  
Coding Region ◽  
Total Rna ◽  
E Coli ◽  
Rat Muscle ◽  
Positional Identity ◽  
Fructose 1,6 Bisphosphatase

Abstract The 1282 bp cDNA of an isoenzyme of fructose-1,6- bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose- 1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 μg of total RNA of rat muscle contains 1.7 × 106 molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 × 104 copies of this message were found per μg total RNA of heart and kidney, respectively.

Bismuth hematoxylin staining of nucleic acids

1995 ◽  
Vol 53 ◽  
pp. 930-931
Author(s):  
A.A. Smith
Keyword(s):  
Nucleic Acids ◽  
Water Soluble ◽  
Metal Salts ◽  
Blue Pigment ◽  
Animal Tissues ◽  
Hematoxylin Staining ◽  
Bismuth Salts ◽  
Ethanesulfonic Acid ◽  
The Stability ◽  
Sodium Bismuthate

Hematoxylin is readily oxidized to the blue pigment hematein. Hematein can complex with a wide variety of metal salts or mordants to form mordant dyes. These dyes stain various components of animal tissues. The component stained depends on the metal in the mordant dye.Alum hematoxylin uses trivalent aluminum ion as a mordant. At extreme dilution, requiring long staining times, alum hematoxylin is a specific stain for nucleic acids. As usually used, alum hematoxylin stains nucleoproteins, staining them nearly as well after the removal of the nucleic acids as before.Bismuth shows a high affinity for nucleic acids. Although all common bismuth salts are insoluble in water, sodium bismuthate reacts with an aqueous solution of hematoxylin to produce hematein and trivalent bismuth ions. The bismuth ions complex with the hematein to form a water-soluble “bismuth hematoxylin.” HEPES (N-[2-hydroxyethyl]piperazine- N'-2-ethanesulfonic acid), 8 mg/ml of solution, greatly increases the amount of bismuth hematoxylin formed. Glycerol, 1/8 v/v, improves the stability of the complex.

The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex

1996 ◽  
Vol 109 (1) ◽  
pp. 209-220 ◽  
Author(s):  
S.L. Lowe ◽  
S.H. Wong ◽  
W. Hong
Keyword(s):  
Amino Acid ◽  
Golgi Complex ◽  
Polyclonal Antibodies ◽  
Brefeldin A ◽  
Amino Acid Identity ◽  
Northern Blotting ◽  
Small Gtpase ◽  
Rat Tissues ◽  
Dependent Manner ◽  
Additional Cell

A rat cDNA clone was isolated which encodes a protein displaying characteristics of a ras-like small GTPase. The deduced amino acid sequence shows the highest amino acid identity (79%) with the Drosophila ARF-like protein 1 (dArl1) among all the known members of the ras-like small GTPase superfamily. The encoded protein was tentatively named rat Arl1 (rArl1). Northern blotting analysis revealed that the rArl1 gene is ubiquitously expressed in rat tissues. Recombinant rArl1 fused to glutathione-S-transferase (GST) to create GST-rArl1 binds GTP-gamma-S in a dose-dependent manner. Polyclonal antibodies raised against two unique rArl1 peptides recognized a 22 kDa protein in total NRK cell lysate. Immunofluorescence microscopy of NRK cells revealed discrete perinuclear labelling that could be competed out by GST-rArl1 but not GST. Examination of 8 additional cell lines revealed a similar labelling, suggesting that the antigen recognised by the antibodies is conserved and widely-expressed. Co-localization experiments in NRK cells with antibodies to mannosidase II and a newly identified cis-Golgi protein, p28, showed that rArl1 is localized to the Golgi complex. When cells were treated with nocodazole, the Golgi complex marked by mannosidase II and p28 was fragmented into punctate structures scattered throughout the cell, in which rArl1 was colocalized. Treatment with brefeldin A (BFA) resulted in the redistribution of rArl1 and mannosidase II into the cytoplasm and endoplasmic reticulum, respectively. The kinetics of the redistribution of rArl1 in response to BFA differ from those of ARF and beta-COP, two components of non-clathrin coated vesicles.

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