University of Western Ontario
--R. B. Zabulionis and D. B. Walden
Retroviral oncogenes were derived from cellular genes during successive infections of the parental viruses into their respective animal hosts (Swanstrom et al., PNAS USA 80:2519, 1983). These oncogenes, unlike their cellular counterparts, do not have introns and consequently are often used as radiolabelled probes in Southern hybridizations when searching for similar sequences. They have been used successfully to detect not only their parental sequence in their respective animal host but also homologous genes in many other species, some of which (e.g. Drosophila, yeast) are quite removed in evolutionary time from the original animal host.
We decided to use a similar strategy (i.e., using viral oncogenes as probes) to detect homologous sequences in maize. It is difficult to obtain hybridization signals above the background when heterologous probes are used on plant genomes. This is particularly evident with maize due to the size and complexity of its genome as well as the abundance of repeated sequences in its genome. These problems can be overcome with the use of several technique modifications and controls (Zabulionis et al., Genome 30:820, 1988). To date we have reported maize sequences homologous to the following animal oncogenes: Ha-ras, Ki-ras, src, myc, myb, and abl (MNL 60:91, 61:72, 62:87).
Last year we reported the detection of maize mRNA transcripts homologous to Ha-ras using Northern blot hybridizations. At that time, "relaxed" hybridization conditions were used (30% formamide, 5.5X SSPE, 56 C) and detected transcripts of 2.8 and 3.2 kilobases.
Since then, more stringent conditions have been used (40% formamide, 3X SSPE, 56 C) in the detection of ras-related transcripts in 5-day-old plumules. The viral Ha-ras probe detected 2 transcripts, 1.6 and 2.0 kilobases in size. The smaller transcripts initially went undetected because of the "relaxed" hybridization conditions and their relative scarcity in comparison to the larger transcripts.
Using the more stringent Northern hybridization conditions, two transcripts in 5-day-old plumules were found to be homologous to Ki-ras. The transcripts are approximately 6kb in size. At the "relaxed" hybridization conditions, Ki-ras also detected the more abundant transcripts (2.8 and 3.2kb) that were detected by Ha-ras.
Sequencing has begun on maize ras-related clones isolated from a cDNA
library made from mRNA of 5-day-old plumules.
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