We reported (MNL 60:91, 1986; 61:72, 1987) on the presence of oncogene-related sequences in maize. In those reports, 32-P labelled probes (v-myb, v-myc, v-src, v-Ki-ras and v-Ha-ras) detected homologous sequences in the maize genome through Southern blot analysis. Under similar hybridization conditions (MNL 61:72, 1987), no fos-related sequences were detected.
The myb-related sequence in maize has been characterized by an alternative method. Paz-Ares et al. (EMBO J. 6:3553, 1987), by transposon tagging and subsequent sequence analysis, have revealed that the c1 gene product in maize is homologous to Drosophila and human c-myb gene products.
Using hybridization conditions and techniques reported previously (MNL 61:72, 1987), preliminary results indicate that maize has sequences homologous to v-abl but not to v-sis. V-sis is the viral homologue of animal platelet-derived growth factor; consequently, it is not surprising that there are no maize sequences homologous to v-sis. V-abl, like v-src, encodes a highly conserved tyrosine kinase domain. It is possible that this domain is the only encoded domain that is common between the animal src genes and the maize src-related sequence. If such were the case, v-abl should bind to the src-related sequence in maize. Southern hybridizations indicate that v-abl does not bind to the src-related sequence in maize. Thus, it appears that the homology between v-src and the related maize sequence encompasses a greater region than the encoded tyrosine kinase domain. However, proof for the extent of homology between the putative maize src gene and animal src genes must await sequence analysis.
We have initiated RNA blot analysis of poly(A)selected RNA from 5-day-old plumule tissue. Total RNA was isolated by the guanidine isothiocyanate method (Methods in Molecular Biology, eds. Davis et al., 1986) and selected twice for poly(A)+ mRNA using mAP paper (Amersham). The poly(A)+ mRNA was electrophoretically separated on glyoxal/DMSO denaturing agarose gels (1.4%) and blotted to Zeta-Probe membrane (BioRad) using 20X SSC as the transfer buffer. The prehybridization buffer consisted of 30% deionized formamide, 5.5X SSPE, 1% SDS, 0.5% "Blotto" (Carnation low fat milk powder), and 500µg/ml of sheared and denatured salmon sperm DNA; incubation temperature was 56 C. The hybridization buffer consisted of the prehybridization buffer plus 20% dextran sulphate and 50ng of radio-labelled oncogene probe; the incubation temperature was 56 C. Washes were done at 60 C in 1X SSPE and 2% SDS.
Figure 1 shows initial Northern blot analysis of poly(A)+ selected maize mRNA probed with Ha-ras.
The Ha-ras detected 2 transcripts (3.2 and 2.8Kb) in young rapidly growing plumule tissue. Ha-ras has been found, in animals, to be expressed in dividing cells during early development. These results provide further evidence that the oncogene-related sequences in maize are genes which may be active during periods of rapid growth.
Work is underway to determine if other maize oncogene-related sequences are transcribed in rapidly growing plumule tissue.
Figure 1. RNA blot of maize poly(A)+ selected RNA hybridized with Ha-ras. Lanes 1-3 have 15, 7.5, and 2µg of mRNA respectively. The size in kilobases and the relative positions of RNA ladder fragments (BRL) are indicated. Larger arrowheads indicate the relative positions of the maize 26s and 17s ribosomal subunits.
R.B. Zabulionis, D.B. Walden, J.D. Procunier* and B.G. Atkinson*
*Department of Zoology
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