More oncogene-related sequences

We reported (MNL 60:91, 1986) on the presence of oncogene-related sequences in maize. In that report, labelled probes (v-Ki-ras and v-src), from Oncor Inc., were used to detect homologous sequences in the maize genome via nick translation and southern hybridization. In this report, we extend the number of oncogenes with related sequences in the maize genome to include v-myc, v-myb, and v-Ha-ras.

Maize DNA was isolated from 5 day old, Oh43 plumules as described by Dellaporta et al. (Molec. Biol. for Plants, pp. 36-37, 1984) and then treated as follows. After resuspension in TE, the DNA was incubated with 250 ug/ml of RNase A at 37 C for one hour, followed by an incubation in proteinase K at 50 C for one hour. The sample was extracted with first phenol/chloroform and then ether followed by dialysis against TE (pH 8.0) for three hours. The DNA was precipitated in 2 M ammonium acetate plus 2 volumes of 95% ethanol and resuspended in TE (pH 8.0) at a concentration of at least 0.4 ug/ul. Digestion of the DNA was carried out overnight with three to four times excess of BamHI or EcoRI using the buffer system recommended by the manufacturer (Boehringer-Mannheim).

E. coli and human EcoRI digested DNA served as controls; all DNA samples were electrophoretically separated on 0.8% agarose gels. Transfer of the DNA to Zeta-Probe (BioRad) charged nylon membrane using 0.4M NAOH as the transfer buffer was undertaken and the blots were rinsed in 2X SSC for 5 minutes and vacuum baked at 80 C for 30 minutes.

Plasmids containing viral oncogene inserts were purchased from American Type Culture Collection. V-myc, v-fos, v-myb and v-Ha-ras inserts were isolated from plasmid pBr322 sequences by cutting with the appropriate restriction endonuclease, electrophoretically separating the fragments on low temperature agarose gels, slicing the gel segment containing the insert out of the rest of the gel, melting it and then purifying the insert through a BioRad RDP column.

Although contamination of inserts with pBR322 was minimal, unlabelled, denatured plasmid was included in each oncogene hybridization reaction to remove any contaminating labelled pBr322. Oncogene inserts were labelled using the Pharmacia oligolabeling kit to a specific activity of approximately 1 x 10 exp 9 cpm/ug.

The prehybridization buffer consisted of 1.5X SSPE, 1% SDS, 0.5% "Blotto" (Carnation low fat milk powder), and 500 ug/ml of sheared and denatured salmon sperm DNA; the incubation temperature was 65 C. Hybridization buffer had 10% dextran sulphate included. Wash buffer was 0.1X SSP 1% SDS at 62 C. Hoeffer's "Hybrid Ease" chambers were used for prehybridizations, hybridizations and washes.

The probes did not detect any homologous sequences in E. coli DNA, confirming an earlier report by Bishop (The Harvey Lectures, Series 78, pp. 137-172, 1982-83).

The v-myc probe detected 2 bands of near identical size (approximately 4.4 kb) in the BamHI digested maize DNA lanes. All previously reported myc-related sequences were detected in the human DNA (c-myc, L-myc, and N-myc). It has been reported (Alitalo et al., PNAS 80:100, 1983) that V-myc contains a 250 bp region with high G-C content (84%) which can lead to spurious hybridization signals. To test for spurious signals, we are removing this region from the insert.

V-myb detected a single sequence in maize of approximately 8 kb. The three myb-related sequences in human EcoRI digested DNA were also detected (Franchini et al., PNAS 80:7385, 1983).

Under the hybridization conditions used, no homologous sequences to v-fos were found in maize.

The v-Ha-ras probe produced several hybridization signals in maize EcoRI or BamHI digested DNA. V-ki-ras had been previously shown (MNL 60:91, 1986) to detect multiple bands in maize DNA. Cross homology was detected between these 2 probes using the hybridization conditions listed above. Although a few maize hybridization signals were unique to each of the two ras probes, most bands were detected by both probes. This complex hybridization pattern between the 2 probes is currently being investigated using high stringency conditions to determine if any of the common sequences are unique to one probe or the other.

These results and those reported last year emphasize the evolutionary conservation of these sequences, thus implying their importance to cell function. Work is underway to determine if the maize sequences are transcribed and if so, in what tissues and under what conditions.

R. Zabulionis, D.B. Walden and J.D. Procunier
 
 


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