Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new -tubulin isoform from sunflower we named -tubulin. -tubulin is the most divergent higher-plant -tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that -tubulin expression is restricted to the male gametophyte. -tubulin mRNA represents 90% of -tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, -tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that -tubulin is specific to this family. Our results suggest that -tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that -tubulin is found in a complex with -tubulin in mature sunflower pollen.     

Modulation of gene expression by α-tocopherol and α-tocopheryl phosphate in THP-1 monocytes

The natural vitamin E analog α-tocopheryl phosphate (αTP) modulates atherosclerotic and inflammatory events more efficiently than the unphosphorylated α-tocopherol (αT). To investigate the molecular mechanisms involved, we have measured plasma levels of αTP and compared the cellular effects of αT and αTP in THP-1 monocytes. THP-1 cell proliferation is slightly increased by αT, whereas it is inhibited by αTP. CD36 surface expression is inhibited by αTP within hours without requiring transport of αTP into cells, suggesting that αTP may bind to CD36 and/or trigger its internalization. As assessed by gene expression microarrays, more genes are regulated by αTP than by αT. Among a set of confirmed genes, the expression of vascular endothelial growth factor is induced by αTP as a result of activating protein kinase B (PKB/Akt) and is associated with increased levels of reactive oxygen species (ROS). Increased Akt(Ser473) phosphorylation and induction of ROS by αTP occur in a wortmannin-sensitive manner, indicating the involvement of phosphatidylinositol kinases. The induction of Akt(Ser473) phosphorylation and ROS production by αTP can be attenuated by αT. It is concluded that αTP and αT influence cell proliferation, ROS production, and Akt(Ser473) phosphorylation in an antagonistic manner, most probably by modulating phosphatidylinositol kinases.     

The primary structure of the human ϵ-globin gene

We describe the complete nucleotide sequence of the human ?-globin gene including 387 nucleotides of 5′ flanking sequence and 301 nucleotides of 3′ flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic β-like globin gene.     

A Single Amino-Acid Substitution at Lysine 40 of an Arabidopsis thaliana α-tubulin Causes Extensive Cell Proliferation and Expansion Defects

Microtubules are highly dynamic cytoskeletal polymers of α/β‐tubulin heterodimers that undergo multiple post‐translational modifications essential for various cellular functions in eukaryotes. The lysine 40 (K40) is largely conserved in α‐tubulins in many eukaryote species, and the post‐translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α‐tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α‐tubulin6 (TUA6) at K40 (TUA6^K40A or TUA6^K40Q), in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S:TUA6^K40A transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α‐ and β‐tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α‐tubulin is critical in maintaining microtubule stability. We also constructed 35S:TUA6^K40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S:TUA6^K40R plants were phenotypically indistinguishable from the wild‐type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α‐tubulins in cell division and expansion.     

Molecular cloning and expression in yeast of 2,3–oxidosqualene– triterpenoid cyclases from Arabidopsis thaliana

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non–photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3–oxidosqualene–triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS–triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, – and –amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUP1 and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.