The maize Tousled-Like Kinase gene family
--Yoon, E; Rivin, C

The Tousled gene of Arabidopsis is essential for normal development of the plant (Roe et al., Cell 75:939, 1993). Mutants in tousled display mild vegetative abnormalities, but are severely impaired in floral development, having a variable, reduced number of floral organs and an unfused gynoecium. Tousled encodes a member of a novel class of serine-threonine kinases. The protein has an N' coiled-coil region, three nuclear localization signals, and a C' catalytic domain. TOUSLED kinase appears to be necessary for the proper maintenance and partitioning of the Arabidopsis shoot apical meristem.

Dr. Tim Helentjaris isolated a partial cDNA with very high homology to the catalytic domain of TOUSLED from developing maize seeds (MNL 69:232, 1995). This cDNA hybridizes to multiple bands on Southern blots, indicating that this gene is a member of a small gene family in maize. This gene and its homologues are the Tousled-Like Kinases, or TLK genes.

We used the maize cDNA from Helentjaris to screen an ear shoot cDNA library and a genomic library. We isolated a slightly longer cDNA of the same gene as Helentjaris' clone. Our cDNA encompasses the entire catalytic domain. In addition, we isolated genomic clones representing four distinct genes, including the one encoding the cDNAs. We have sequenced portions of the catalytic domains of each of these genes. They are strikingly similar, with over 92% identity to each other and over 85% identity to TOUSLED at the amino acid level. There is also conservation of gene structure. Three of the genes contain introns in the same locations as each other and as Tousled (Figure 1). The sequence of the 3' untranslated region and the most 3' intron is also quite conserved in maize. The fourth gene lacks introns.

We have detected very low level expression of at least one TLK gene. Northern blots show a transcript of between 2.5 and 3.0 kb. This size is consistent with the size of the Tousled transcript. Due to the extremely high homology of the maize genes, we have not determined whether this signal represents a single transcript, or the expression of more than one TLK gene. However, both cDNAs isolated correspond to the gene which lacks introns, indicating that this gene is expressed in both developing inflorescences and seeds.

Tousled is a single gene in Arabidopsis , but the TLK genes of maize form a small gene family. The sequenced portions of all four genes contain open reading frames encoding potentially functional kinases. We are interested in the function of these kinases in maize development and in whether the individual members of this gene family have redundant or distinctive roles. We are currently analyzing the non-catalytic domains of these genes. These regions are likely to be more divergent and may allow specific regulation of each of the TLK proteins. We have also initiated a TUSC reverse genetics search to obtain mutants in these loci.

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