Molecular analysis of holocentric centromeres of Luzula species.

Luzula spp, like the rest of the members of the Juncaceae family, have holocentric chromosomes. Using the rice 155-bp centromeric tandem repeat sequence (RCS2) as a probe, we have isolated and characterized a 178-bp tandem sequence repeat (LCS1) from Luzula nivea. The LCS1 sequence is present in all Luzula species tested so far (except L. pilosa) and like other satellite repeats found in heterochromatin, the cytosine residues are methylated within the LCS1 repeats. Using fluorescent in situ hybridization (FISH) experiments we have shown that there are at least 5 large clusters of LCS1 sequences distributed at heterochromatin regions along each of the 12 chromosomes of L. nivea. We have shown that a centromeric antibody Skp1 co-localizes with these heterochromatin regions and with the LCS1 sequences. This suggests that the LCS1 sequences are part of regions which function as centromeres on these holocentric chromosomes. Furthermore, using the BrdU assay to identify replication sites, we have shown that these heterochromatin sites containing LCS1 associate when being replicated in root interphase nuclei. Our results also show premeiotic chromosome association during anther development as indicated by single-copy BAC in situ and the presence of fewer LCS1 containing heterochromatin sites in these cells.

Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis.

Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. Genes encoding Ran-like proteins have been isolated from different higher plant species. Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46-1 cell cycle mutant in yeast cells. The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran-binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (RanBPs) specific for Ran-GTP. Here, the characterization of the first Ran-binding proteins from higher plants is reported. The yeast two-hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana. The deduced amino acid sequences of the At-RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran-binding domains. Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C-terminal domain (-DEDDDL) of Ran proteins in addition to the presence of an intact Ran-binding domain. In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins. Finally, in good agreement with their proposed biological function, the At-Ran and the At-RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.

Characterization of membrane-bound small GTP-binding proteins from Nicotiana tabacum.

We have cloned nine cDNAs encoding small GTP-binding proteins from leaf cDNA libraries of tobacco (Nicotiana tabacum). These cDNAs encode distinct proteins (22-25 kD) that display different levels of identity with members of the mammalian Rab family: Nt-Rab6 with Rab6 (83%), Nt-Rab7a-c with Rab7 (63-70%), and Nt-Rab11a-e with Rab11 (53-69%). Functionally important regions of these proteins, including the "effector binding" domain, the C-terminal Cys residues for membrane attachment, and the four regions involved in GTP-binding and hydrolysis, are highly conserved. Northern and western blot analyses show that these genes are expressed, although at slightly different levels, in all plant tissues examined. We demonstrate that the plant Rab5, Rab6, and Rab11 proteins, similar to their mammalian and yeast counterparts, are tightly bound to membranes and that they exhibit different solubilization characteristics. Furthermore, we show that the yeast GTPase-activating protein Gyp6, shown to be specifically required to control the GTP hydrolysis of the yeast Ypt6 protein, could interact with tobacco GTP-binding proteins. It increases in vitro the GTP hydrolysis rate of the wild-type Nt-Rab7 protein. In addition, it also increases, at different levels, the GTP hydrolysis rates of a Nt-Rab7m protein with a Rab6 effector domain and of two other chimaeric Nt-Rab6/Nt-Rab7 proteins. However, it does not interact with the wild-type Nt-Rab6 protein, which is most similar to the yeast Ypt6 protein.

Molecular characterization and expression of a tobacco histone H1 cDNA.

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steady-state mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.

Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein.

Mutations in which the onset of mitosis is uncoupled from the completion of DNA replication has recently been described. Characterization of these mutants led to the identification of Pim1/Spi1 in fission yeast and RCC1/Ran proteins in mammalian cells. Their Saccharomyces cerevisae homologues, the MTR1/CNR1 proteins, appear to be involved in controlling RNA metabolism and transport. Here the isolation and partial characterization of plant cDNA clones which encode proteins homologous to the mammalian/fission yeast/budding yeast Ran/Spi/CNR proteins are reported. Higher plants appear to contain more than one gene per haploid genome which codes for Ran proteins. These genes are expressed in different plant tissues, including root tips and stems, known to contain mitotically active cells. The tobacco Ran-like proteins, like their mammalian and yeast homologues, are soluble proteins which are found in the cytoplasm and in the nucleus. In addition, it has been shown that overexpression of the tobacco Nt-Ran-A1 cDNA suppressed the phenotype of the temperature-sensitive fission yeast pim1-46 mutant. These results suggest that the plant Ran genes can be functionally equivalent to the mammalian/fission yeast/budding yeast Ran/Spi/CNR genes and that they may play a role: (i) in maintaining a coordinated cell cycle; (ii) in controlling RNA metabolism and transport in higher plants; and/or (iii) in protein import into the nucleus.