Oncogene-related sequences in maize

The acutely oncogenic retroviruses are capable of rapidly inducing a variety of leukemias and sarcomas in appropriate animal hosts. The retroviral genes responsible for these transformations arose from sequences of the respective host. These transforming genes are termed "oncogenes" whereas their apparently normal, cellular counterparts are referred to as "proto-oncogenes". Proto-oncogenes are transcribed and translated in young, growing tissues and are considered essential for growth and differentiation (Pardee, A. B. et al., Cellular oncogenes, growth factors, and cellular growth control, pp. 21-29, 1985).

Oncogene related sequences (proto-oncogenes) have been reported in many vertebrate species as well as in several invertebrate (fruit fly and nematode) and lower plant species (yeast and slime mold). This report extends the species to have oncogene related sequences to include Zea mays.

Nuclei were isolated from 5 day old Oh43 maize seedlings as described by Luthe and Quatrano (Plant Physiol. 65:305, 1980). Lysis of nuclei and isolation of DNA was carried out according to Zimmer and Newton (Maize for Biol. Res., pp. 165-168, 1982). The DNA was digested overnight with 4 times excess of ApaI, HindIII, or EcoRI using the buffer system recommended by the manufacturer (Boehringer-Mannheim). Digested maize DNA was size fractionated on 0.8% agarose gels. To serve as positive controls, avian and human EcoRl digested DNA was run on each gel. DNA from the gels was blotted to nitrocellulose membranes (BioRad, 0.45 um) using standard Southern transfer procedures. Viral Kirstein ras oncogene (v-Ki-ras) and viral src oncogene (v-src) were used to probe the blots. Prehybridization and hybridization procedures were adapted from Wahl et al. (PNAS 76:3683, 1979). To increase the likelihood of hybridization of the probes to the maize DNA, 30% formamide was used instead of 50% formamide and the hybridization temperature was 37 C.

DNA from human and avian tissue that was used as a positive control produced hybridization patterns similar to those previously reported (McGarth, J. F., et al., Nature 304:501, 1983; Gibbs, C. P., et al., J. Virol. 53:19, 1985). Both oncogene probes detected homologous sequences in maize DNA. The blots probed with v-Ki-ras produced a complex hybridization pattern (at least 4 bands in each lane of digested maize DNA - ApaI, HindIII, and EcoRI). This suggests that there is more than one gene in maize homologous to v-Ki-ras. (It is unlikely that a single gene would have multiple ApaI, HindIII, and EcoRI restriction sites.) ApaI digested DNA showed 4 bands at approximately 7.5, 6.2, 4.3 and 2.6 kb. HindIII and EcoRI digested maize DNA had 4 bands between 4.3 and 1.0 kb. These results are similar to those reported from animal studies: vertebrate genomes contain several ras proto-oncogenes. The v-src hybridized blot had 1 prominent band in each lane of maize digested DNA: ApaI, 6.6kb; HindIII, 1.9 kb: and EcoRI, 2.8 kb. This suggests that the nuclear genome of maize contains a single sequence homologous to v-src. From hybridization conditions, it was determined that these maize sequences have at least 70% homology to the viral oncogene probes (Howley, P M., et al., J. Biol. Chem. 254:4876, 1979).

R. Zabulionis, D. B. Walden and B. G. Atkinson
 
 


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