rnase  (Worthington Biochemical)

Journal: Genes & Development

Article Title: Localization-dependent translation requires a functional interaction between the 5? and 3? ends of oskar mRNA

doi:

Figure Lengend Snippet: p50 binds both to the 5′ end of osk ) was incubated and UV cross-linked to proteins in the oocyte extract in the absence of competitor RNA (lane 1 ). Competitions were carried out with a 100-fold excess of either repressor fragment (rep, lane 2 ), Eco RI– Bgl II fragment containing the 5′ activator in sense orientation (5′/act-s, lane 3), or the same Eco RI– Bgl II fragment in antisense orientation (5′/act-as, lane 4 ). Immunoprecipitation of proteins UV cross-linked to radiolabeled repressor fragment (lane 5 ) by anti-Bruno antiserum (lane 6 ) or preimmune serum (lane 7 ). Simultaneous binding of p50 and Bruno was tested by treating with RNase A only after the immunoprecipitation. (Lane 8 ) Anti-Bruno antiserum; (lane 9 ) preimmune serum. We noted that in some cases (as shown here) the intensity of the upper band of the p50 doublet was reduced after coprecipitation. The locations of the 5′ and 3′ competitors in the osk transcript are indicated below. The radiolabeled probe is indicated by an asterisk. The reverse experiment, with activator element RNA as a radioactive probe, and cold activator and repressor RNA elements as unlabeled competitors, yielded the same result with respect to p50 (data not shown).

Article Snippet: Fifty micrograms of RNase A and 4 units of RNase T1 (Worthington Biochemical) were used.

Techniques: Incubation, Activated Clotting Time Assay, Immunoprecipitation, Binding Assay

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Liquid Chromatography with Mass Spectroscopy, Chromatography, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Mass Spectrometry, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation

doi: 10.1093/nar/gkab272

Figure Lengend Snippet: Mt FRS activity. ( A ) Aminoacylation of unmodified M. tuberculosis tRNA Phe . The 3′- 32 P-labeled tRNA transcript was charged with L-Phe in the presence of purified Mt FRS. The extent of aminoacylation was analyzed according to Varshney et al. ( 37 ). Prior to polyacrylamide gel electrophoresis, tRNA and Phe-tRNA were digested with RNase T1 to yield 3′- 32 P-labeled CCACCA and CCACCA-Phe, respectively. (+) and (-) indicate the presence and the absence of the enzyme. (B–D) Synthesis of the Phe-AMP intermediate by Mt FRS in the presence of unmodified M. tuberculosis tRNA Phe ( B ), E. coli tRNA Phe ( C ) and yeast tRNA Phe ( D ). Phe-[ 32 P]AMP synthesis was monitored by thin layer chromatography. Phe-AMP and ATP denote 32 P-labeled compounds. F – full reaction; -E, -tRNA and -Phe – control reactions lacking the enzyme, tRNA or amino acid.

Article Snippet: Prior to electrophoresis, on a 10% acid/7 M urea polyacrylamide gel, 10 pmoles of uncharged and aminoacylated tRNA (Phe-tRNAPhe) were completely digested with 2 U of RNase T1 (Worthington) in 10 mM sodium acetate (pH 4.5).

Techniques: Activity Assay, Labeling, Purification, Polyacrylamide Gel Electrophoresis, Thin Layer Chromatography

Journal: Nature methods

Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

doi: 10.1038/nmeth.1705

Figure Lengend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

Article Snippet: Resuspended nuclei were digested (30 min; 33°C) with either (i) DNase I (protease- and RNase-free; Worthington;10 units/107 cells in 100 μl PB plus 0.5 mM CaCl2 ), or (ii) Hae III (Invitrogen; 1000 units/107 cells), or (iii) Hind III (New England Biolabs; 1000 units/107 cells) in PB; reactions were stopped by adding EDTA to 2.5 mM and cooling in iced water.

Techniques: Purification, Mass Spectrometry, Activity Assay, Fractionation