608 Nucleic Acids Research, Vol. 20,
A
new
No.
.=; 1992
3
Oxford University Press
3'-terminus for Xenopus laevis 28S ribosomal RNA
Murray N.Schnare and Michael W.Gray* Program in Evolutionary Biology, Canadian Institute for Advanced Research, Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada Submitted December 13, 1991 An SI nuclease protection assay has previously been used to determine the position of the 3'-terminus of Xenopus laevis 28S rRNA (1). However, primary sequence comparisons (2) and secondary structure modelling (3) suggest that X. kaevis 28S rRNA should contain an additional six nucleotides at its 3'-end. We have now obtained data that support this prediction. A poly(A)- RNA fraction from X. laevis was used as a source of 28S rRNA, which was purified as described (4), 3'-endlabelled, and subjected to sequence analysis by the chemical degradation procedure of Peattie (5). An autoradiogram of the sequencing gel is presented in Fig. 1, with the deduced sequence being numbered from the 3'-end. The data demonstrate that X. laevis 28S rRNA contains six additional 3'-terminal nucleotides that had not been revealed previously by S1 nuclease protection experiments (the horizontal arrow indicates the position of the S1 nuclease-determined 3'-end). This discrepancy can be explained by the fact that of the six additional 3'-terminal nucleotides reported here, five are U residues, and it is known that rU:dA hybrids are particularly sensitive to hydrolysis by S1 nuclease (6). The RNA sequence data presented here also verify the presence of an A residue at position 14 from the 3'-end. This residue is absent from one previously determined DNA sequence of this region (7) but is present in two others (1, 8).
ACKNOWLEDGEMENTS We thank M.E.MacKay and C.B.Lazier for a gift of X. laevis poly(A)- RNA and the Canadian Institute for Advanced Research for salary support (M.W.G.). This work was supported by an operating grant (MT-1 1212) from the Medical Research Council of Canada to M.W.G.
REFERENCES 1. Sollner-Webb,B. and Reeder,R.H. (1979) Cell 18, 485-499. 2. Kominami,R., Mishima,Y., Urano,Y., Sakai,M. and Muramatsu,M. (1982) Nucleic Acids Res. 10, 1963-1979. 3. Gutell,R.R., Schnare,M.N. and Gray,M.W. (1990) Nucleic Acids Res. 18 (Suppi.), 2319-2330.
*
To whom correspondence should be addressed
EMBL accession
no.
X62823
4. Schnare,M.N., Heinonen,T.Y.K., Young,P.G. and Gray,M.W. (1986) J. Biol. Chem. 261, 5187-5193. 5. Peattie,D.A. (1979) Proc. Natl. Acad. Sci. USA 76, 1760-1764. 6. Miller,K.G. and Sollner-Webb,B. (1981) Cell 27, 165-174. 7. Ware,V.C., Tague,B.W., Clark,C.G., Gourse,R.L., Brand,R.C. and Gerbi, S.A. (1983) Nucleic Acids Res. 11, 7795-7817. 8. Moss,T., Boseley,P.G. and Birnstiel,M.L. (1980) Nucleic Acids Res. 8, 467-485.
A G
I
. a=
A
new
No.
.=; 1992
3
Oxford University Press
3'-terminus for Xenopus laevis 28S ribosomal RNA
Murray N.Schnare and Michael W.Gray* Program in Evolutionary Biology, Canadian Institute for Advanced Research, Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada Submitted December 13, 1991 An SI nuclease protection assay has previously been used to determine the position of the 3'-terminus of Xenopus laevis 28S rRNA (1). However, primary sequence comparisons (2) and secondary structure modelling (3) suggest that X. kaevis 28S rRNA should contain an additional six nucleotides at its 3'-end. We have now obtained data that support this prediction. A poly(A)- RNA fraction from X. laevis was used as a source of 28S rRNA, which was purified as described (4), 3'-endlabelled, and subjected to sequence analysis by the chemical degradation procedure of Peattie (5). An autoradiogram of the sequencing gel is presented in Fig. 1, with the deduced sequence being numbered from the 3'-end. The data demonstrate that X. laevis 28S rRNA contains six additional 3'-terminal nucleotides that had not been revealed previously by S1 nuclease protection experiments (the horizontal arrow indicates the position of the S1 nuclease-determined 3'-end). This discrepancy can be explained by the fact that of the six additional 3'-terminal nucleotides reported here, five are U residues, and it is known that rU:dA hybrids are particularly sensitive to hydrolysis by S1 nuclease (6). The RNA sequence data presented here also verify the presence of an A residue at position 14 from the 3'-end. This residue is absent from one previously determined DNA sequence of this region (7) but is present in two others (1, 8).
ACKNOWLEDGEMENTS We thank M.E.MacKay and C.B.Lazier for a gift of X. laevis poly(A)- RNA and the Canadian Institute for Advanced Research for salary support (M.W.G.). This work was supported by an operating grant (MT-1 1212) from the Medical Research Council of Canada to M.W.G.
REFERENCES 1. Sollner-Webb,B. and Reeder,R.H. (1979) Cell 18, 485-499. 2. Kominami,R., Mishima,Y., Urano,Y., Sakai,M. and Muramatsu,M. (1982) Nucleic Acids Res. 10, 1963-1979. 3. Gutell,R.R., Schnare,M.N. and Gray,M.W. (1990) Nucleic Acids Res. 18 (Suppi.), 2319-2330.
*
To whom correspondence should be addressed
EMBL accession
no.
X62823
4. Schnare,M.N., Heinonen,T.Y.K., Young,P.G. and Gray,M.W. (1986) J. Biol. Chem. 261, 5187-5193. 5. Peattie,D.A. (1979) Proc. Natl. Acad. Sci. USA 76, 1760-1764. 6. Miller,K.G. and Sollner-Webb,B. (1981) Cell 27, 165-174. 7. Ware,V.C., Tague,B.W., Clark,C.G., Gourse,R.L., Brand,R.C. and Gerbi, S.A. (1983) Nucleic Acids Res. 11, 7795-7817. 8. Moss,T., Boseley,P.G. and Birnstiel,M.L. (1980) Nucleic Acids Res. 8, 467-485.
A G
I
. a=
