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A Factor in Serum and Amniotic Fluid Is a Substrate for the Trna-Modifying

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A Factor in Serum and Amniotic Fluid Is a Substrate for the Trna-Modifying Corrections Proc. Natl. Acad. Sci. USA 76 (1979) 5411 Correction. In the article "Fibronectin and the multiple in- Correction. In the article "A factor in serum and amniotic fluid teraction model for platelet-collagen adhesion" by Samuel A. is a substrate for the tRNA-modifying enzyme tRNA-guanine Santoro and Leon W. Cunningham, which appeared in the June transferase" by Jon R. Katze and Walter R. Farkas, which ap- 1979 issue of Proc. Natl. Acad. Sci. USA (76,2644-2648), de- peared in the July 1979 issue of Proc. Natl. Acad. Sci. USA (76, fective printing obscured some words on p. 2645 in some copies. 3271-3275), the authors request that the following correction The first complete paragraph in the second column on that page be noted. On p. 3271, in the second column, line 6 from the should read as follows: bottom, "Sephadex G-100" should be "Sephadex G-10." Effect of Chymotrypsin Treatment of Platelets. The effect of platelet surface alteration by protease treatment was exam- ined. Chymotrypsin was selected because it does not induce the release reaction and initiate aggregation as do other proteases such as trypsin and thrombin (28). Treatment of platelet sus- Correction. In the article "Colony-stimulating factor (CSF) pensions with 30 jug of chymotrypsin per ml decreased the radioimmunoassay: Detection of a CSF subclass stimulating extent of adhesion of platelets to collagen. The decrease was macrophage production" by E. Richard Stanley, which ap- dependent upon the time of enzyme treatment (Fig. 3). Thus, peared in the June 1979 issue of Proc. Natl. Acad. Sci. USA (76, the adhesion of platelets to collagen is likely mediated by 2969-2973), the author requests that the following corrections platelet surface proteins. be noted. On page 2970, line 28 of the left-hand column, "50,000 cpm/ng" should read "250,000 cpm/ng." In line 39 of the same column, "0.2 ng, 24 units" should read "40 pg, 6 units." In Fig. 3, the symbols for two sources should be inter- Correction. In the article "Sequence relationships among de- changed. C57BL pokeweed mitogen mouse spleen CM should fective interfering influenza viral RNAs" by Alan R. Davis and be represented by * and C57BL mouse endotoxin lung CM by Debi P. Nayak, which appeared in the July 1979 issue of Proc. * As a consequence, in Table 1 the values for percentage col- Natl. Acad. Sci. USA (76,3092-3096), the authors request that ony-stimulating activity detectable by RIA should also be in- the following correction be noted in the legends of Figs. 2 and terchanged for these two sources. The percentage for pokeweed 3: "second dimension, bottom to top" and not "top to bottom" mitogen spleen CM should be 0.4 and the percentage for en- as stated. dotoxin lung CM should be 4. Downloaded by guest on September 29, 2021 Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3271-3275, July 1979 Biochemistry A factor in serum and amniotic fluid is a substrate for the tRNA- modifying enzyme tRNA-guanine transferase (modified nucleoside Q/tRNAAsP/tRNAHis/posttranscriptional modification) JON R. KATZEt AND WALTER R. FARKASt tDepartment of Microbiology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163; and tDepartment of Medical Biology, University of Tennessee Center for the Health Sciences, Knoxville, Tennessee 37920 Communicated by William A. Arnold, April 30, 1979 ABSTRACT Q factor, a substance found in animal serum replaced by guanine is the guanine residue in the first position that enables cultured mammalian cells (L-M) to produce tRNA of the anticodon (N. K. Howes and W. R. Farkas, unpublished containing queuine (the base of "nucleoside Q", queuosine), has data) and similar results have been obtained with the Esche- been purified to homogeneity from bovine amniotic fluid. Q richia coli and Drosophila enzymes (refs. 13 and 14; K. B. Ja- factor causes the appearance of Q-containing tRNAASP in the L-M cells cultivated in serum-free medium, and this was used cobson and W. R. Farkas, unpublished data). These findings as an assay to monitor the purification of Q factor. Q factor is suggest that tRNA-guanine transferase participates in the a competitive inhibitor of guanine for rabbit reticulocyte substitution. The enzyme is thought to remove guanine from tRNA-guanine transferase, with a K1 of 4.5 X 10-8 M. Q factor the first position of the anticodon and to replace it with queuine is inactivated in both the IrM cell and tRNA-guanine transferase or a precursor of it (9, 15-18); however, in the absence of this assays by treatment with periodate or cyanogen bromide, both substrate, it catalyzes the previously noted (11, 12) exchange of which react with queuine. In L-M cells, nearly complete conversion of Q-free to Q-containing tRNAAsP is observed within of guanine for guanine. 24 hr after addition of pure Q factor to the medium; actinomycin Katze (19) has described a serum component, designated Q D, cycloheximide, and cycloleucine, inhibitors of RNA synthesis, factor, which enables cells in tissue culture to synthesize protein synthesis, and nucleic acid methylation, respectively, [Q+]tRNAs from [Q-]tRNA. The properties of Q factor are do not inhibit this conversion. The product of the reaction, consistent with it being either a substrate for the guanine- catalyzed by pure rabbit reticulocyte tRNA-guanine transferase, transferring enzyme or a precursor that is converted into a between Q factor and rabbit reticulocyte Q-free tRNAHiS is substrate. In the present communication, we describe the pu- chromatographically indistinguishable from Q-containing rification of Q factor to homogeneity from bovine amniotic tRNAHiS. fluid and show that it is a substrate for the reticulocyte tRNA- The hypermodified guanosine derivatives queuosine (Q) and guanine transferase. modified queuosine (Q*) are found in the first position of the anticodons of tRNATYr, tRNAHis, tRNAAsn, and tRNAAP, which MATERIAL AND METHODS form a set in that they accommodate the codons NAU (1, 2). Yeast tRNA was obtained from Miles, [3H]guanine [specific Queuosine is 7-1[(cis-4,5-dihydroxy-2-cyclopenten-1-yl)- activity, 1 Ci/mmol (1 Ci = 3.7 X 101 becquerels)] from ICN, amino]-methyl}-7-deazaguanosine (1); Q* designates derivatives [3H]histidine (1 Ci/mmol) and [14C]histidine (0.24 Ci/mmol) of Q having either a mannose or a galactose residue at the 4 or from Schwarz/Mann, and protein assay solution and globulin 5 position of the cyclopentenediol (2). The Q-containing tRNAs standard from Bio-Rad.-Buffers, salts, and L aminoacids were from tumors or tissue cultured cells often show differences in reagents grade. Rabbit liver guanine aminohydrolase (1.0 chromatographic patterns when compared with the same unit/mg) was from Sigma and bovine amniotic fluid was from tRNAs from normal animal tissue such as liver (refs. 3-7 and Pel-Freez. Protein was assayed as described (20), as was the refs. therein). This phenomenon is explained by variations in guanylation reaction (9). tRNA-guanine transferase was puri- the extent of the Q modifications. The tRNA isoaccepting fied to homogeneity from rabbit erythrocytes as described (21), species that contain Q, [Q+]tRNA, are eluted from RPC-5 and as was the in vio Q factor activity in L-M cells cultured in the Aminex A-28 columns earlier than are their unmodified absence of serum (19). counterparts, [Q-]tRNA, in which the queuine position is oc- Purification of Q factor. Bovine amniotic fluid (517 ml) was cupied by guanine (3-10). The additional positive charge of Q subjected to ultrafiltration (Amicon hollow fiber cartridge, type is sufficient to explain this chromatographic difference. HiP10); the retentate was washed with two 50-ml portions of tRNA-guanine transferase (formerly called guanine inser- water. The entire ultrafiltrate (600 ml) was added to a column tase), described by Farkas and coworkers in mammalian re- (5 X 88 cm) of Sephadex G-100 in water; elution was with water ticulocytes (11, 12), catalyzes the exchange of guanine into until the effluent showed negligible absorbance at 280 nm, with tRNAs of the NAU codon set. In homologous enzyme-tRNA 0.02 M HC1 until the effluent showed no material absorbing at systems, [Q-]tRNAs, but not [Q+]tRNAs, are substrates for the 280 nm, and finally with 0.45 M NH40H. Significant Q-factor enzyme (9, 10, 13, 14), which excises a specific base by cleaving activity was found only in the 0.02 M HC1 eluate. The active the N-glycosyl bond and replaces that base with guanine. fraction was lyophilized, redissolved in methanol, and subjected Studies with the pure reticulocyte enzyme show that the base Abbreviations: Q, queuosine; [Q+]tRNA, tRNA that contains Q in the The publication costs of this article were defrayed in part by page first position of the anticodon; [Q-]tRNA, the unmodified or precursor charge payment. This article must therefore be hereby marked "ad- form of [Q+]tRNA, which contains guanosine instead of Q in the first vertisement" in accordance with 18 U. S. C. §1734 solely to indicate position of the anticodon. In this study, Q is used generically to desig- this fact. nate Q and its saccharide derivative Q*. 3271 3272 Biochemistry: Katze and Farkas Proc. Natl. Acad. Sci. USA 76 (1979) to preparative thin-layer chromatography on cellulose plates aqueous phases were pooled, and the RNA was recovered by (Eastman Chromogram type 13225), with the following solvent ethanol precipitation. The precipitate was dissolved in 0.05 M systems in sequence: A, 3% ammonium formate; B, isopropa- sodium acetate, pH 4.5/5 mM 2-mercaptoethanol/5 mM nol/2% ammonium acetate, 1:1 (vol/vol) (22); C, 1-butanol/ EDTA/0.01 M MgCl2/0.3 M NaCl and added to a DEAE- glacial acetic acid/H20, 4:1:1 (vol/vol) (22).
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