MILAN, ITALY
Università degli Studi di Milano

The ra1-154 mutation is caused by K53 deletion in the zinc finger domain

— Cassani, E; Landoni, M; Curiale, S; Cerino Badone, F; Panzeri, D; Pilu, R

In order to investigate molecular changes in the ra1-154 allele (previously described by Pilu et al., MNL 78:39–40), we cloned it by PCR and then performed sequence analysis; specific primers were designed on the basis of the Ra1 sequence cloned by Martienssen and Vollbrecht, 2001 (patent number WO0190343).

Alignment of the Ra1 and ra1-154 sequences revealed that the mutant carries an AGG deletion in the coding region. This small deletion causes the loss of the lysine residue at position 53 in the putative C₂H₂ zinc-finger domain (Fig. 1). Cosegregation analysis performed using allele-specific primers showed that ra1-154 deletion was always associated with the mutant phenotype. Furthermore, RT-PCR analysis showed that Ra1 and ra1-154 have the same expression level in the inflorescence tissue. On the whole, these data suggest that the K53 deletion could be responsible for the loss of the protein’s function, most probably due to its inability to build the zinc-finger domain. This is the first evidence of single amino acid deletion in the zinc-finger domain that knocks out the function of the RA1 protein. This result strongly suggests that the RA1 protein functions by acting as a DNA-binding protein likely involved in transcriptional regulation.

Fig. 1. Alignment of the deduced RA1 and RA1-154 proteins. The AGG deletion in the ra1-154 gene causes the loss of lys 53 in the putative C₂H₂ zinc-finger domain (indicated by the thick line).

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