Spindle microtubules seemed to be most resistant to disorganization

Spindle microtubules seemed to be most resistant to disorganization. a genomic clone encoding -tubulin from a liverwort (Bryophyta), genomic DNA. The decided nucleotide sequence was used to design PCR primers to isolate the entire genomic DNA of the -tubulin gene by means of the vector-annealing PCR method. Sequencing of both strands of the PCR fragments revealed that this -tubulin of was encoded by a gene encompassing 3582 bp and was intervened by 10 introns. Connecting 11 exons revealed that there was an open reading frame of 1428 bp that encoded a protein of 475 amino acid residues. Throughout the process of -tubulin gene identification, we encountered no evidence, such as amplification of genomic fragment of more than one size or obvious sequence polymorphism among the isolated clones after vector-annealing PCR amplification, of the presence of more than one -tubulin gene in the haploid genome of genomic DNA was digested with restriction enzymes and probed with a DNA probe of the moss -tubulin. Only the bands of expected sizes from the sequences of isolated genes were detected (data not shown). These data indicate that there are no other genes homologous with -tubulin in genome, as is the case with other lower land plants, such as the fern (Fuchs et al., 1993) and the moss -tubulin with the sequences of other known -tubulins. First, it was immediately apparent that -tubulin belonged to the conventional group of Bucetin -tubulins, because it shared at least 67.5% amino acid identity with other known conventional -tubulins. On the other hand, -tubulin was 39% identical to the -tubulinClike protein Tub4p, one of the unconventional -tubulins. The -tubulin gene product is usually highly conserved among land plants (Physique 1). The Bucetin results of our amino acid comparison among herb -tubulins is usually shown in Physique 1B. The -tubulin showed 89.2 to 97.7% amino acid identity to those of other land plants (-tubulin was 74.7% to that of and 69.3% to that of the fission yeast and F3 other herb -tubulins. The deduced amino acid sequence of the -tubulin is usually shown in the top row. Amino acid sequences of herb -tubulins were aligned, and amino acids identical to those of the -tubulin are indicated by dashes. Dots indicate Bucetin gaps introduced for the alignment. The inferred recognition epitope of the G9 antibody is usually underlined. (At), (Nt), (Zm), (Cj), (Pp), (Ap), (Cr), (Sp), -tubulin (Horio et al., 1999), to detect -tubulin homologs in various species of bryophytes. The epitope detected by the G9 antibody has been studied and narrowed down to amino acid residues 97 to 111 of -tubulin (GGGAGNNWANGYSHA; our unpublished data). This region is almost completely conserved among known herb -tubulins (Physique 1B, underlined) and is fairly well conserved in -tubulin (11 of 15 amino residues are identical). In immunoblots of extracts from sporophytes of is usually 53,359 D. Comparable results were obtained in extracts of sporophytes from another bryophyte, (Physique 2B). G9 has been used successfully for immunofluorescence staining of seed plants (Ovenchkina and Oakley, 2001). The -tubulin of Arabidopsis expressed in the fission yeast has been detected by immunoblot analysis and immunofluorescence staining using G9 (our unpublished data). These facts indicated that this epitope detected by G9 in a variety of bryophytes most likely is usually -tubulin. Open in a separate window Physique 2. Immunoblot Analysis of G9 Antibody in Protein Extracts from Sporogenous Tissue of the Bryophytes and (Brown and Lemmon, 1988; Shimamura et al., 1998). Except for the RMS, these MTOCs have never been seen in seed plants. Reproductive cells generally lack unique cortical microtubule systems. To ascertain the occurrence of -tubulin in these MTOC sites, we examined the localization of G9 anti–tubulin cross-reactive materials in putative MTOCs in bryophytes Marchantia polymorpha During mitosis in polyplastidic cells of marchantialean liverworts, plastids do not serve as MTOCs. Instead, POs, which arise de novo outside of the nuclear envelope, are the foci of a.