A DNA insertion within Adh1 is transcribed

Last year H. P. Doring, M. Motto, F. Salamini and P. Starlinger (MNL 56:40, 1982) described an Adh1 mutant they recovered from plants homozygous for bz2-m, containing Ac. The mutant, 2F11, has no ADH1, activity and presumably contains a Ds element affecting the Adh1 gene. In collaboration with Starlinger's lab, we have been studying 2F11 to determine: (1) if it responds to Ac, (2) whether there is an insertion at or near the Adh1 gene, and (3) if there is transcription of the Adh1 gene. This note will describe the RNA data; the genetic and restriction mapping data will be described elsewhere.

I compared poly-adenylated ADH1-RNA from roots of 2F11 and a standard line, 1S, after seedlings were subjected to 12 hours of anaerobic stress. The RNA samples were electrophoresed on a denaturing formaldehyde gel, transferred to nitrocellulose and probed with the Adh1 cDNA clone, pZML84 (Figure 1). The ADH1-RNA of 2F11 is present to the same extent as in 1S, but the 2F11 RNA is 2 kb larger. The increase in size of the message is compatible with the results of our Southern data that demonstrate the presence of a 2 kb insert within the coding region (to be published elsewhere). In addition to the larger ADH1-RNA of 2F11, a minor amount of normal size message (1650 bp) is also present. The normal size message is probably not ADH2 because of the stringency of the hybridization and wash conditions (Tm -20 C and Tm -10 C, respectively). To determine if transcription starts and stops at the normal positions, 2F11 ADH1-RNA was probed with 3' and 5' fragments of the Adh1 genomic clone, pB428. The results of this experiment showed that the novel transcript has Adh1 coding sequence surrounding the insertion.

Our interest in 2F11 goes beyond the Adh1 gene. 2F11 displays a mild knotted phenotype that is linked to Adh1 (M. Freeling, pers. comm.). The knotted gene, Kn1, is on chromosome 1 and is the nearest known neighbor to Adh1 (< 0.1 mu). We intend to ask if the knotted phenotype is due to a position effect of the insertion at Adh1, or if Kn1 is at the insertional site within the Adh1 coding sequence (seems unlikely?) or, something more complicated.

Figure 1. 2F11 and 1S seedlings were subjected to 12 hours of anaerobic stress. One ug of poly-adenylated RNA from 1S and 2F11 anaerobic roots were electrophoresed on a formaldehyde % agarose gel. The gel was transferred to nitrocellulose and probed with an Adh1 cDNA clone. BMV RNA was used as a size standard.

Sarah Hake

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