Preparation, molecular weight, base composition, and secondary structure of giant nuclear ribonucleic acid

Holmes, David S.; Bonner, James

doi:10.1021/bi00736a023

Cited by 371 publications

(178 citation statements)

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“…Plasmid rescue: Purification of genomic DNA was after Holmes and Bonner (1973) and plasmid rescue was according to Pirrotta (1986). Genomic DNA was digested with BamHI, XbaI, or double digested with XbaI and SpeI, diluted, and ligated.…”

Section: Methodsmentioning

confidence: 99%

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

et al. 2005

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Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 59-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient ''jumpstarting'' scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila. O NE of the main goals of modern genetics is to link the many thousands of genes identified through the sequencing of whole genomes of model organisms to gene function. The most powerful technique for this purpose so far has been transgenesis with mobile elements. This technique is a means to disrupt, overexpress, or misexpress single genes to identify expression patterns and also to characterize genetic pathways and their interactions. One of the main advantages of insertional mutagenesis over the classical method of chemical mutagenesis is the ease with which the targeted gene can be identified, since it carries an inserted tag.The P element was the first mobile element that enabled germ-line transformation of an insect species (Rubin and Spradling 1982). Since then, thousands of single P-element insertions causing lethality, semilethality, sterility, semisterility, and visible phenotypes have been created and analyzed in Drosophila (Cooley et al.

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Section: Methodsmentioning

confidence: 99%

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

et al. 2005

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“…The nucleated erythroid cell suspension (0.9 ml) was added to 100 ml of lysing solution (15), which contained the nuclease inhibitors used in the proteinase K procedure. Phenol/chloroform (100 ml) was added immediately and subsequent isolation steps were the same as dcescribed by Holmes and Bonner (15) (17). The 10 cm gels were run for 6'A hr at 5 mA per gel.…”

Section: Methodsmentioning

confidence: 99%

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Kwan¹,

Wood²,

Lingrel³

1977

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

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Nucleated erythroid cells were incubated for 10 min in the presence of [5-Hluridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% ofthe radioactively-labeled total cellular RNA applied to the column annealed to globin comlementary DNA cellulose. (27 Ci/mmol), were added and the incubation continued for an additional 10 min. After the incubation, the cells were chilled on ice, collected by centrifugation, washed once with 10 ml of cold (40) isotonic saline, and resuspended in 2 ml of cold saline. Isolation of RNA Proteinase K Procedure. The cell suspension (0.9 ml) was added dropwise with stirring to 50 ml of a solution (12) containing 30 mM Tris-HCI, (pH 7.4), 100 mM NaCl, 5 mM EDTA, 3% sodium dodecyl sulfate (NaDodSO4), heparin (150 yg/ml) dextran sulfate (100 ,g/ml), 3 mM each of 2' and 3' AMP, CMP, and UMP, and 300 ,ug/ml of proteinase K. The proteinase K was autodigested in the above buffer, without NaDodSO4, for 10 min at 350 prior to use. The cell suspension was incubated for 30 min at 350. After the incubation, 50 ml of the above buffer including NaDodSO4, but without proteinase K were added, followed by 100 ml of buffer-saturated, redistilled phenol and chloroform (1:1, vol/vol). The mixture was stirred at room temperature for 15 min. The aqueous phase was collected by centrifugation at 5000 X g for 5 min at 200.The interphase was re-extracted with extraction buffer minus proteinase K and the combined aqueous phases were re-extracted twice with phenol/chloroform and once with chloroform alone. The aqueous solution was adjusted to 0.3 M NaCl and the nucleic acids were precipitated with 2 volumes of absolute ethanol at -200. After allowing the mixture to stand overnight at -20°, the nucleic acids were collected by centrifugation and washed three times with 70% ethanol. The nucleic acids were immediately dissolved with stirring at 30, in 10 ml of a solution containing 10 mM sodium acetate (pH 5.2), 3 mM MgCI2, heparin (200 ,g/ml), polyvinyl sulfate (30 ,ug/ml), dextran sulfate (200 Ag/ml), 3 mM each of 2' and 3' AMP, CMP, and UMP and 40,g/ml of DNase (Worthington RNase-free) which had been treated with iodoacetate (13). The mixture was stirred at 100 for 20-25 min during which time the viscosity became markedly reduced. The solution was then made 2% in NaDodSO4, 5 mM in EDTA, and 300 ,tg/ml of proteinase K was added to digest the DNase. After a 10 min incubation at 300, sodium acetate (pH 5.2) was added to give a final concentration of 50 mM and an equal volume of phenol/chloroform was then added and the mixture stirred for 5 min at 25°. The aqueous layer was collected, re-extracted first with phenol/chloroform and then twice with chloroform, and the RNA precipitated as described above. The RNA pellet was washed with 70% ethanol, dissolved in water and stored at -700...

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“…Globin mRNA was prepared from isolated reticulocyte polysomes by phenol extraction [31] and repeated oligo (dT)-cellulose chromatography [32]. ['4C]Phe-tRNA was prepared as described previously [33].…”

Section: Preparation Of Globin Mrna and [14c]phe-trnamentioning

confidence: 99%

The mechanism of the protein‐synthesis elongation cycle in eukaryotes

Nilsson

Nygård

1986

European Journal of Biochemistry

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The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pretranslocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1 -catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 . GTP . aminoacyl-tRNA . ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1-and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.The translational elongation in eukaryotes is mediated through the two elongation factors EF-1 and EF-2 and involves a cyclic series of reactions by which the amino acids are added to the growing polypeptide chain [l, 21. Each cycle can be divided into three basic reaction steps. In the first step cognate aminoacyl-tRNAs are brought to the A-site of the mRNA-programmed ribosome in the form of a ternary complex with EF-1 and GTP [l, 21. During this reaction, GTP is hydrolysed to GDP and inorganic phosphate [3]. This step is followed by the transfer of the nascent peptide in the ribosomal P-site to the newly attached aminoacyl-tRNA [l, 21. Finally, elongation factor EF-2 promotes the translocation of the elongated peptidyl-tRNA from the A-site to the P-site under the hydrolysis of GTP [4]. A common GTPase center on the ribosome, involving the 5s rRNA . L5 complex, has been suggested to participate in both the EF-1-and EF-2-dependent GTP hydrolysis [5].The two elongation factors bind to the ribosome at identical or partially overlapping binding sites [6 -91. Elongation factor EF-1 associates exclusively with post-translocation ribosomes, i. e. ribosomes having their peptidyl-tRNA in the P-site, whereas EF-2 binds only to pre-translocation ribosomes, i.e. ribosomes carrying the peptidyl-tRNA in the Asite [6, 71. We have previously demonstrated that EF-2 forms two types of ribosomal complexes [lo]. In the presence of nonhydrolysable GTP analogues a high-...

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Preparation, molecular weight, base composition, and secondary structure of giant nuclear ribonucleic acid

Cited by 371 publications

References 39 publications

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

The mechanism of the protein‐synthesis elongation cycle in eukaryotes

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