In trangenic rice, α- and β-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression

The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5′UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice β-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant β-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice α-tubulin and two rice β-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5′UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Generation of a database containing discordant intron positions in eukaryotic genes (MIDB)

MOTIVATION: Intron sliding is the relocation of intron-exon boundaries over short distances and is often also referred to as intron slippage or intron migration or intron drift. We have generated a database containing discordant intron positions in homologous genes (MIDB--Mismatched Intron DataBase). Discordant intron positions are those that are either closely located in homologous genes (within a window of 10 nucleotides) or an intron position that is present in one gene but not in any of its homologs. The MIDB database aims at systematically collecting information about mismatched introns in the genes from GenBank and organizing it into a form useful for understanding the genomics and dynamics of introns thereby helping understand the evolution of genes. RESULTS: Intron displacement or sliding is critically important for explaining the present distribution of introns among orthologous and paralogous genes. MIDB allows examining of intron movements and allows mapping of intron positions from homologous proteins onto a single sequence. The database is of potential use for molecular biologists in general and for researchers who are interested in gene evolution and eukaryotic gene structure. Partial analysis of this database allowed us to identify a few putative cases of intron sliding. AVAILABILITY: http://intron.bic.nus.edu.sg/midb/midb.html     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.     

A large plant beta-tubulin family with minimal C-terminal variation but differences in expression

Tubulins, as the major structural component of microtubules (MT), are highly conserved throughout the entire eukaryotic kingdom. They consist of alpha/beta heterodimers. Both monomers, at least in multicellular organisms, are encoded by gene families. In higher plants up to eight beta-tubulin isotypes, mostly differing in their very C-termini, have been described. These variable beta-tubulin C-termini have been discussed in the context of functional microtubule diversity. However, in plants, in contrast to vertebrates, functional isotype specificity remains yet to be demonstrated. Unlike higher plants, unicellular green algae in general do not exhibit isotypic variations. The moss Physcomitrella patens is a phylogenetic intermediate between higher plants and green algae. We isolated six beta-tubulin genes from Physcomitrella, named PpTub1 to 6. We show that the exon/intron structure, with the exception of one additional intron in PpTub6, is identical with that of higher plants, and that some members of the family are differentially expressed. Moreover, we find that all Physcomitrella isotypes are highly conserved and, most strikingly, are almost identical within their C-terminal amino acids (aa). This evolutionary ancient and large beta-tubulin gene family without significant isotypic sequence variation points to a role of differential regulation in the evolution of plant tubulin isotypes.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.     

Evolutionary analysis of three gibberellin oxidase genes in rice, Arabidopsis, and soybean

GAs are plant hormones that play fundamental roles in plant growth and development. GA2ox, GA3ox, and GA20ox are three key enzymes in GA biosynthesis. These enzymes belong to the 2OG-Fe (II) oxygenase superfamily and are independently encoded by different gene families. To date, genome-wide comparative analyses of GA oxidases in plant species have not been thoroughly carried out. In the present work, 61 GA oxidase family genes from rice (Oryza sativa), Arabidopsis, and soybean (Glycine max) were identified and a full study of these genes including phylogenetic tree construction, gene structure, gene family expansion and analysis of functional motifs was performed. Based on phylogeny, most of the GA oxidases were divided into four subgroups that reflected functional classifications. Intron/intron average length of GA oxidase genes in rice analysis revealed that GA oxidase genes in rice experienced substantial evolutionary divergence. Segmental duplication events were mainly found in soybean genome. However, in rice and Arabidopsis, no single expansion pattern exhibited dominance, indicating that GA oxidase genes from these species might have been subjected to a more complex evolutionary mechanism. In addition, special functional motifs were discovered in GA20ox, GA3ox, and GA2ox, which suggested that different functional motifs are associated with differences in protein function. Taken together our results suggest that GA oxidase family genes have undergone divergent evolutionary routes, especially at the monocot-dicot split, with dynamic evolution occurring in Arabidopsis thaliana and soybean.     

Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species.

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant beta-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different beta-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Convergent intron loss of MRP1 in Drosophila and mosquito species

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.     

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.     

The <Emphasis Type="Italic">Endo-β-Mannanase</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis</Emphasis>, rice,and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4-d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.     

ExInt: an Exon/Intron database

The Exon/Intron (ExInt) database incorporates information on the exon/intron structure of eukaryotic genes. Features in the database include: intron nucleotide sequence, amino acid sequence of the corresponding protein, position of the introns at the amino acid level and intron phase. From ExInt, we have also generated four additional databases each with ExInt entries containing predicted introns, introns experimentally defined, organelle introns or nuclear introns. ExInt is accessible through a retrieval system with pointers to GenBank. The database can be searched by keywords, locus name, NID, accession number or length of the protein. ExInt is freely accessible at http://intron.bic.nus.edu.sg/exint/exint.html     

Isolation and sequence analysis of a beta-tubulin gene from Aspergillus flavus and its use as a selectable marker

An altered beta-tubulin gene that confers resistance to benomyl [whose active ingredient is 2-(methoxycarbonylamino)benzimidazole (MBC)] was isolated from a DNA library of Aspergillus flavus and used as a selectable marker for transformation. The beta-tubulin gene was cloned into a plasmid vector containing the pyr-4 gene of Neurospora crassa, and transformants were selected either for uracil prototrophy or MBC resistance. Transformants selected for uracil prototrophy were of three phenotypic classes: sensitive, intermediate, and resistant to MBC. Transforming DNA appeared to integrate at several sites in the genome, with the more resistant phenotypes having more copies of the altered beta-tubulin gene than the sensitive and intermediate phenotypes. Transformants were also selected on medium containing MBC. The average frequency of transformation (1 to 3 transformants per micrograms of transforming DNA) was lower than that obtained by selection for uracil prototrophy, presumably because of failure to select transformants that contained few copies of the altered beta-tubulin gene. The sequence of the beta-tubulin gene was determined and compared with the published sequence of the benA gene of A. nidulans; the beta-tubulin gene was found to be highly conserved between the two Aspergillus species. Notable differences were that the beta-tubulin gene of A. flavus lacks intron 6 present in benA and has an additional leucine at position 148. This is the first gene sequence reported from an aflatoxin-producing fungus and adds to the growing body of knowledge of the beta-tubulin genes and their use as selectable markers for transformation of filamentous fungi.