To study the Myb gene family in maize, we isolated and sequenced Myb gene sequences derived from a maize BAC genomic library (ZMMBBb; http://www.genome.clemson.edu/orders/ lib_desc/ZMMBBb.html). Myb-containing BAC clones were detected using as probe the radiolabeled fragment of a maize Myb gene (P1-wr) cDNA covering the R2R3 Myb domain. We detected 143 Myb-hybridizing clones from two filters (NSF B73E, serial# 009687; and NSF B73F, serial# 010186) containing 36,864 BAC clones covering 2 genome equivalents. The BAC clones were initially sequenced using a degenerate primer (5’-GAKGYCSGGSCGVAGGTAGTT-3’) complementary to sequences within exon 2 of 78 maize Myb EST sequences (sequence data kindly provided by Dr. Erich Grotewold, Ohio State University). The sequences obtained using the degenerate primer were used to design new primers specific for each clone for further sequencing in the upstream and downstream directions. Further rounds of sequencing followed by specific primer design continued until complete Myb domain sequences were obtained. Using this primer walking strategy, we obtained sequences from 44 BAC clones and detected 31 unique Myb genes (redundancy = 30%). This figure is consistent with previous data indicating that the maize genome contains more than 80 expressed Myb genes (Rabinowicz et al., Genetics 153:427-444, 1999). Obviously, the Myb gene family has expanded broadly during the evolution of flowering plants.
The sequences of the 31 unique Myb genes have been deposited into NCBI. The accession numbers are AF474115 ~ AF474124 (with complete R2R3 domains), AF470072 ~ AF470092 (with partial R2R3 domains due to sequencing difficulties). The maize BAC addresses were also deposited into the Maize Mapping Project (http://www.maizemap.org). It is hoped that the maize Myb gene sequences may assist in the mapping of individual Myb genes, and eventual identification of the specific metabolic pathways and phenotypic traits controlled by each Myb gene.
`Using the Myb genomic sequences, we attempted to identify conserved
regulatory motifs in non-coding regions with the assistance of computational
tools. First, we constructed a phylogenetic tree and identified clades
of closely-related Myb genes. Then, motif-finding tools (MEME and Macaw)
were applied to search for motifs within each clade. Besides the Myb domain
itself, some highly conserved motifs were identified in the C-terminal
coding regions of various diverse Myb proteins. In the non-coding regions,
conserved motifs were identified only in the clade containing the maize
p1 and p2 genes, and orthologs from sorghum and rice. Within
this clade, a highly conserved pattern of TATA-box, Transcription Start
Site sequences, and 5’ UTR CA-box was found. Otherwise, no significant
regulatory motifs were detected in non-coding regions of other Myb genes.
Our results suggest that it will be difficult to directly identify gene
regulatory motifs in non-coding regions using only existing computational
techniques. Possibly, the identification of coexpressed genes using microarray
analysis will assist in the identification of common regulatory elements.
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