Gene, 110 (1992) 263-264 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
263
GENE 06255
Brief Notes Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA (Recombinant DNA; blocks of G + C-rich sequences; myc oncogene; filter hybridization)
Steven Dooley, Uwe Firber, Cornelius Welter, Birgit Theisinger and Nikolaus Blin Institut far Humangenetik. Universitiit des Saarlandes. 6650 Homburg/Saar (F.R.G.) Received by S.T. Case: 14 April 1991 Accepted: 19 May 1991 Received at publishers: 1 Novemb~,r 1991
SUMMARY
In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb.specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.
Although expression of the myb oncogene in most cases is associated with hematological disorders, several recent observations suggested specific activity in some human solid tumor cells (e.g., Griffing and Baylin, 1985; Kiefer et al., 1987). The considerable variation in reported transcript sizes and some discrepancies with controls in our brain tumor studies prompted us to investigate the validity of the data in more detail. Unexpectedly, genomic or cDNA probes of human myb or mouse (pHM~!.6, pMM49) (Dozier et al., 1986; Gonda et al., 1985) displayed notable cross-hybridization to 28S rRNA in Northern blots when using total cellular RNA (Fig. IA). This was confirmed by hybridizing a myb clone to DNA encoding 28S rRNA (rDNA), even after stringent washing conditions (0.1 x SSC, 65°C) (Fig. 1B). A computer search using the Correspondenceto: Dr. S. Dooley, Institut fOr Humang,~:netik,Universitat des Saarlandes, 6650 Homburg/Saar (F.R.G.) Tel. (49)6841-166602; Fax (49)6841-166600. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; myb, avian myeloblastosis oncogene; myc, ~'dan myelocytomatosis oncogene; nt, nucleotide(s); oligo, oligodeoxyribmucleotide; rDNA, DNA encoding rRNA; rRNA, ribosomal RNA; S!IC, 150 mM NaCI/ 15 mM Na3" citrate pH 7.6.
'BSA' program (Devereux et al., 1984) indicated similar blocks of G + C-rich sequences (human rDNA, nt 31123234 and 4309-4346; human myb, nt 1-119 and 30333070). To overcome this problem, we have used a myb oligo (mRNA nt 147-189; N-terminal aa 11-24, supplied by Oncogene Science, Manhasset, NY) as a probe with total cellular RNA preparations. The myb-overexpressing cell line Molt-4 (Westin et al., 1982) showed a clear signal (3.5 kb), other cells remained negative (Fig. 2A). The quality of the Northern bl,ot was tested by hybridizing the blot to a 28S rDNA oligo (Fig. 2B) (Barbu and Dautry, 1989). Finally, Molt-4 RN,A was hybridized with priM2.6 and, expectedly, yielded two bands (3.5 and 4.8 kb). Our data clearly indicate the potential pitfalls of unexpected crosshybridization also reported for G + C-rich blocks in the c-myc sequence (Shilo and Weinberg, 1981) and show the usefulness of defined oligo probes. They also suggest a careful re-evaluation of some reports of myb oncogene expression.
ACKNOWLEDGEMENTS Supported in part by a DFG grant (B1166/3-3).
264 1
REFERENCES
3
A 1 -28S
1
2
2
3
A
3
B 6.64.8-
g
II Ill
-28S
kb Fig. 1.
Fig. 2.
Fig. 1. Hybridization signals of human myb recombinant DNA (priM2.6). (Panel A) Northern blot with total RNA from the glioblastoma cell lines (lanes: 1, HERO; 2, A 172; 3, T405). (Panel B) Southern blot with recombinant plasmid DNA. Lanes: 1, EcoRI fragment of the mouse rDNA, pUCMrl0 (Grummt, 1981); 2, SalI fragment of the sea urchin rDNA, pLy4 (Blin et al., 1979); 3, the recombinant plasmid of the human c-myb, priM2.6 (Dozier et al., 1986) including covalent (supercoiled) and nicked circles and the linear form indicated as I, II and Ill. For Northerns, isolated RNA were separated on 1% agarose-0.66 M formaldehyde gel, blotted to Genescreen membranes and hybridized with the nick-translated myb plasmid. For Southerns, recombinant DNA from the species indicated were separated on 1% agarose gels, transferred to Genescreen and hybridized as above. Fig. 2. Northern-blot hybridization of total RNA from glioblastoma cell lines (lanes: I, HeRo; 2, A172; 3, Molt-4) with (Panel A) a myb oligo, and (Panel B)a 28S rDNA oligo (Barbu and Dautry, 1989). RNA electrophoresis and blotting were as described in Fig, 1, hybridization was performed with oligos end-labeled by polynueleotide kinase.
Barbu, V. and Dautry, F.: Northern blot normalization with a 28S rRNA oligonucleotide probe. Nucleic Acids Res. 17 (1989) 7115. Blin, N., Sperrazza, J.M., Wilson, F.E., Bieber, D.G., Mickel, F.S. and Stafford, D.W.: Organization of the ribosomal RNA gene cluster in Lytechinus variegatus. J. Biol. Chem. 254 (1979) 2716-2721. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programms for the VAX. Nucleic Acids Res. 12 0984) 387-395. Dozier, C., Walbaum, S., Lcprince, D. and Stehelin, D.: EcoRl RFLP linked to the human myb gene. Nucleic Acids Res. 14 (1986) 1928. Gonda, TJ., Gough, N., Dunn, A.R. and de Blaquiere, J.: Nucleotide sequence of eDNA clones of the murine c-myb proto-oncogene. EMBO J. 4 (1985) 2003-2008. Griffing, C.A. and Baylin, S.B.: Expression of the c.myb oncogene in human small cell lung carcinoma. Cancer Res. 45 (1985) 272-275. Grummt, !.: Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vice. Prec. Natl. Acad. Sci. USA 78 (1981) 727-731. Kiefer, E., Bepler, G., Kubasch, M. and Havemann, K.: Amplification and expression of protooncogenes in human small cell lung cancer cell lines. Cancer Res. 47 (1987) 6236-6242. Shilo, B. and Weinberg, R.A.: DNA sequences homologous to vertebrate oncogenes are conserved in Dmsophgamelanogaster. Prec. Natl. Acad. Sci. USA 78 (198t) 6789-6792. Westin, E,H., Gallo, R.C., Arya, S.K., Eva, A., Souza, L.M., Baluda, M.A., Aaronson, S.A. and Wong-Staal, F.: Differential expression of the amy gene in human hematopoietic cells. Prec. Natl. Acad. Sci. USA 79 (1982) 2194-2198.
263
GENE 06255
Brief Notes Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA (Recombinant DNA; blocks of G + C-rich sequences; myc oncogene; filter hybridization)
Steven Dooley, Uwe Firber, Cornelius Welter, Birgit Theisinger and Nikolaus Blin Institut far Humangenetik. Universitiit des Saarlandes. 6650 Homburg/Saar (F.R.G.) Received by S.T. Case: 14 April 1991 Accepted: 19 May 1991 Received at publishers: 1 Novemb~,r 1991
SUMMARY
In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb.specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.
Although expression of the myb oncogene in most cases is associated with hematological disorders, several recent observations suggested specific activity in some human solid tumor cells (e.g., Griffing and Baylin, 1985; Kiefer et al., 1987). The considerable variation in reported transcript sizes and some discrepancies with controls in our brain tumor studies prompted us to investigate the validity of the data in more detail. Unexpectedly, genomic or cDNA probes of human myb or mouse (pHM~!.6, pMM49) (Dozier et al., 1986; Gonda et al., 1985) displayed notable cross-hybridization to 28S rRNA in Northern blots when using total cellular RNA (Fig. IA). This was confirmed by hybridizing a myb clone to DNA encoding 28S rRNA (rDNA), even after stringent washing conditions (0.1 x SSC, 65°C) (Fig. 1B). A computer search using the Correspondenceto: Dr. S. Dooley, Institut fOr Humang,~:netik,Universitat des Saarlandes, 6650 Homburg/Saar (F.R.G.) Tel. (49)6841-166602; Fax (49)6841-166600. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; myb, avian myeloblastosis oncogene; myc, ~'dan myelocytomatosis oncogene; nt, nucleotide(s); oligo, oligodeoxyribmucleotide; rDNA, DNA encoding rRNA; rRNA, ribosomal RNA; S!IC, 150 mM NaCI/ 15 mM Na3" citrate pH 7.6.
'BSA' program (Devereux et al., 1984) indicated similar blocks of G + C-rich sequences (human rDNA, nt 31123234 and 4309-4346; human myb, nt 1-119 and 30333070). To overcome this problem, we have used a myb oligo (mRNA nt 147-189; N-terminal aa 11-24, supplied by Oncogene Science, Manhasset, NY) as a probe with total cellular RNA preparations. The myb-overexpressing cell line Molt-4 (Westin et al., 1982) showed a clear signal (3.5 kb), other cells remained negative (Fig. 2A). The quality of the Northern bl,ot was tested by hybridizing the blot to a 28S rDNA oligo (Fig. 2B) (Barbu and Dautry, 1989). Finally, Molt-4 RN,A was hybridized with priM2.6 and, expectedly, yielded two bands (3.5 and 4.8 kb). Our data clearly indicate the potential pitfalls of unexpected crosshybridization also reported for G + C-rich blocks in the c-myc sequence (Shilo and Weinberg, 1981) and show the usefulness of defined oligo probes. They also suggest a careful re-evaluation of some reports of myb oncogene expression.
ACKNOWLEDGEMENTS Supported in part by a DFG grant (B1166/3-3).
264 1
REFERENCES
3
A 1 -28S
1
2
2
3
A
3
B 6.64.8-
g
II Ill
-28S
kb Fig. 1.
Fig. 2.
Fig. 1. Hybridization signals of human myb recombinant DNA (priM2.6). (Panel A) Northern blot with total RNA from the glioblastoma cell lines (lanes: 1, HERO; 2, A 172; 3, T405). (Panel B) Southern blot with recombinant plasmid DNA. Lanes: 1, EcoRI fragment of the mouse rDNA, pUCMrl0 (Grummt, 1981); 2, SalI fragment of the sea urchin rDNA, pLy4 (Blin et al., 1979); 3, the recombinant plasmid of the human c-myb, priM2.6 (Dozier et al., 1986) including covalent (supercoiled) and nicked circles and the linear form indicated as I, II and Ill. For Northerns, isolated RNA were separated on 1% agarose-0.66 M formaldehyde gel, blotted to Genescreen membranes and hybridized with the nick-translated myb plasmid. For Southerns, recombinant DNA from the species indicated were separated on 1% agarose gels, transferred to Genescreen and hybridized as above. Fig. 2. Northern-blot hybridization of total RNA from glioblastoma cell lines (lanes: I, HeRo; 2, A172; 3, Molt-4) with (Panel A) a myb oligo, and (Panel B)a 28S rDNA oligo (Barbu and Dautry, 1989). RNA electrophoresis and blotting were as described in Fig, 1, hybridization was performed with oligos end-labeled by polynueleotide kinase.
Barbu, V. and Dautry, F.: Northern blot normalization with a 28S rRNA oligonucleotide probe. Nucleic Acids Res. 17 (1989) 7115. Blin, N., Sperrazza, J.M., Wilson, F.E., Bieber, D.G., Mickel, F.S. and Stafford, D.W.: Organization of the ribosomal RNA gene cluster in Lytechinus variegatus. J. Biol. Chem. 254 (1979) 2716-2721. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programms for the VAX. Nucleic Acids Res. 12 0984) 387-395. Dozier, C., Walbaum, S., Lcprince, D. and Stehelin, D.: EcoRl RFLP linked to the human myb gene. Nucleic Acids Res. 14 (1986) 1928. Gonda, TJ., Gough, N., Dunn, A.R. and de Blaquiere, J.: Nucleotide sequence of eDNA clones of the murine c-myb proto-oncogene. EMBO J. 4 (1985) 2003-2008. Griffing, C.A. and Baylin, S.B.: Expression of the c.myb oncogene in human small cell lung carcinoma. Cancer Res. 45 (1985) 272-275. Grummt, !.: Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vice. Prec. Natl. Acad. Sci. USA 78 (1981) 727-731. Kiefer, E., Bepler, G., Kubasch, M. and Havemann, K.: Amplification and expression of protooncogenes in human small cell lung cancer cell lines. Cancer Res. 47 (1987) 6236-6242. Shilo, B. and Weinberg, R.A.: DNA sequences homologous to vertebrate oncogenes are conserved in Dmsophgamelanogaster. Prec. Natl. Acad. Sci. USA 78 (198t) 6789-6792. Westin, E,H., Gallo, R.C., Arya, S.K., Eva, A., Souza, L.M., Baluda, M.A., Aaronson, S.A. and Wong-Staal, F.: Differential expression of the amy gene in human hematopoietic cells. Prec. Natl. Acad. Sci. USA 79 (1982) 2194-2198.
