Isolation and characterisation of two pollen-specific LIM domain protein cDNAs from Nicotiana tabacum

A PCR-generated DNA probe covering the region corresponding to the two cont iguous LIM domain sequences from the sunflower PLIM-1 cDNA was used to scre en a cDNA library prepared from poly-A(+) RNA isolated from mature Nicotian a tabacum pollen. Two clones were isolated and found to have unique but clo sely related nucleotide sequences NTPLIM1a and NTPLIM1b. Homology searches revealed the presence of two LIM domain repeats in each clone. Detailed spa tial and temporal northern blot analysis showed that the NTPLIM transcripts were present specifically in mature and germinating pollen and were detect able immediately before the asymmetrical division pollen mitosis I, reachin g a maximal level in mature pollen. Southern blot analysis revealed the NTP LIM1a and NTPLIM1b genes to be single-copy and members of a small gene fami ly in the N. tabacum genome. Analysis of a genomic clone encoding NTPLIM1a revealed that the gene is split by Four introns ranging from 79 to 922 bp i n length. The high levels of developmentally regulated expression specifica lly in pollen suggests an important role for these proteins in pollen matur ation and/or function.