Leader Intron Research Articles

Wx alleles in rice: relationship with apparent amylose content of starch and a possible role in rice domestication

The Waxy locus of rice is a highly polymorphic region embedded with microsatellite repeats in the 5'UTR leader intron 1 region, 23-bp duplication (wx motif) in exon 2, SNPs in exons 4, 6 and 10, p-Sine-r2 element in intron 1 and TnR-1 element in inton 13. Of the 80 polymorphic sites detected on the Wx gene, 24 are located in p-Sine-r2 and TnR-1 elements, revealing a higher substitution rate of bases in these two regions. All the cultivars with chalky endosperm had the 5'-AGTTATA-3' haplotype in intron 1 and 'A' to 'G' substitution at ?497 in exon 4. The AAC of starch from grains of all the accessions showed strong correlation (r=0.967) with GBSS-I activity in the grains. Based on the polymorphic sites of the Waxy locus and the GBSS-I activities, six allelic variants were defined which included wx, Wxop, Wxb, Wxin, Wxa2 and Wxa1, respectively, corresponded to glutinous, very low, low, intermediate, highII and highI amylose classes. Phylogenetic tree developed from alignment matrix of nucleotide sequences of the Waxy locus identified wx, Wxb and Wxin alleles with japonica lineage of Oryza sativa and the Wxop, Wxa2 and Wxa1 with indica lineage.

Single nucleotide polymorphisms in GBBSI and SSIIa genes in relation to starch physicochemical properties in selected rice (Oryza sativa L.) varieties

Single nucleotide polymorphisms in GBBSI and SSIIa genes in relation to starch physicochemical properties in selected rice (Oryza sativa L.) varieties

Natural selection supports escape from concerted evolution of a recently duplicated CEACAM1 paralog in the ruminant CEA gene family

Concerted evolution is often observed in multigene families such as the CEA gene family. As a result, sequence similarity of paralogous genes is significantly higher than expected from their evolutionary distance. Gene conversion, a “copy paste” DNA repair mechanism that transfers sequences from one gene to another and homologous recombination are drivers of concerted evolution. Nevertheless, some gene family members escape concerted evolution and acquire sufficient sequence differences that orthologous genes can be assigned in descendant species. Reasons why some gene family members can escape while others are captured by concerted evolution are poorly understood. By analyzing the entire CEA gene family in cattle (Bos taurus) we identified a member (CEACAM32) that was created by gene duplication and cooption of a unique transmembrane domain exon in the most recent ancestor of ruminants. CEACAM32 shows a unique, testis-specific expression pattern. Phylogenetic analysis indicated that CEACAM32 is not involved in concerted evolution of CEACAM1 paralogs in ruminants. However, analysis of gene conversion events revealed that CEACAM32 is subject to gene conversion but remarkably, these events are found in the leader exon and intron sequences but not in exons coding for the Ig-like domains. These findings suggest that natural selection hinders gene conversion affecting protein sequences of the mature protein and thereby support escape of CEACAM32 from concerted evolution.

Isolation and characterization of Ulva prolifera actin1 gene and function verification of the 5′ flanking region as a strong promoter

ABSTRACTUlva prolifera is a green macroalgae with an extremely high growth rate that can accumulate biomass with considerable protein content. To set up an available seaweed expression system, a prior step is to isolate endogenous and strong constitutive promoters. For this reason, the full-length genomic actin1 gene from U. prolifera (Upactin1) was cloned and its 5′ flanking sequence was obtained by genome walking. The Upactin1 open reading frame consisted of 1134 nucleotides encoding 377 amino acid residues. Besides 4 exons and 3 introns in the coding region, an extra leader intron was identified in the 5′ untranslated region. According to quantitative GUS assays based on transient expression, the promoter activity of the Upactin1 5′ flanking region was found to be several times higher than that of the widely-used cauliflower mosaic virus 35S (CaMV35S) in all tested species of Ulva. In addition, precise deletion of the leader intron led to a significant decrease of promoter strength in U. prolifera, and almost entire loss of strength in U. linza and U. pertusa. To our knowledge, this is the first report to prove function of a leader intron in algae. The 5′ flanking region of Upactin1 was shown to be a much stronger promoter than the foreign CaMV35S, and its activity was highly dependent on the presence of the leader intron. We propose that the Upactin1 promoter could serve as an endogenous and strong constitutive element for genetic engineering of U. prolifera.

Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.

Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Sequence polymorphism in the Waxy locus and its relationship with apparent amylose content of endosperm starch in cultivars of rice (Oryza sativa L.) from Northeast India

The Northeast India, being at the confluence of Indo-Malayan and Indo-Chinese biogeographic realms, represents one of the world “biodiversity hotspots”. With a rich diversity of cultivars, the rice gene pool of the region shows marked variations in various quality traits including aroma and starch quality. Since starch quality is primarily determined by its apparent amylose content and that the Waxy locus controls much of the variation in apparent amylose content, the linkage between sequence polymorphism in the Waxy locus and the apparent amylose content assumes significance. Our investigations on the relationship between SNPs in Waxy locus and variations in AAC of starch varieties of rice from North East India clearly reveal that in accessions having the sequence AGTTAT in 5′ leader intron splice site, “Ex6C” allele was associated with starches having 0–2% AAC and “Ex6A” allele was associated with starches having between 2 and 12% AAC. Based on combination of “G/T” polymorphism in 5′ leader intron splice site, length polymorphism in exon 2 and “A/C” polymorphism in exon 6, the Waxy locus of rice can have four allelic forms viz. “Int1T-Ex2(23 bp)2-Ex6C”, “Int1T-Ex2(23 bp)1-Ex6A”, “Int1G-Ex2(23 bp)1-Ex6C” and “Int1G-Ex2(23 bp)1-Ex6A” which correlate specifically with “waxy”, “very low”, “low/intermediate” and “high amylo-” cultivars, respectively. Phylogenetic tree on alignment matrix of nucleotide sequences of the p-SINEI element of the Waxy locus clustered the upland and japonica cultivars separately from the lowland and indica cultivars, thereby establishing the role of transposons in the Waxy locus in differentiation of upland/japonica and lowland/indica cultivars.

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters.

Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

The 5'UTR Intron of Arabidopsis GGT1 Aminotransferase Enhances Promoter Activity by Recruiting RNA Polymerase II.

Photorespiration is essential for the detoxification of glycolate and recycling of carbon to the Calvin Benson Bassham cycle. Enzymes participating in the pathway have been identified, and investigations now focus on the regulation of photorespiration by transporters and metabolites. However, regulation of photorespiration on the gene level has not been intensively studied. Here, we show that maximum transcript abundance of Glu:glyoxylate aminotransferase 1 (GGT1) is regulated by intron-mediated enhancement (IME) of the 5' leader intron rather than by regulatory elements in the 5' upstream region. The intron is rich in CT-stretches and contains the motif TGTGATTTG that is highly similar to the IME-related motif TTNGATYTG. The GGT1 intron also confers leaf-specific expression of foreign promoters. Quantitative PCR analysis and GUS activity measurements revealed that IME of the GGT1 5'UTR intron is controlled on the transcriptional level. IME by the GGT1 5'UTR intron was at least 2-fold. Chromatin immunoprecipitation experiments showed that the abundance of RNA polymerase II binding to the intron-less construct is reduced.

Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv. 9311 and its mutant with extremely high amylose phenotype (9311eha) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Significant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron 5′ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efficiencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not significant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

Development of lentiviral vectors for transient and stable protein overexpression in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) production

BackgroundRecombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5′UTR (5′ untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. ResultsBy combining the human cytomegalovirus (CMV) or the elongation factor 1α (EF-1α) promoters along with different 5′UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5′UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines. ConclusionIn the present work, LVs containing different promoters and 5′UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.

Characterization of differential expression and leader intron function of Arabidopsis atTOC159 homologous genes by transgenic plants.

BackgroundAccurate import of thousands of nuclear-encoded proteins is an important step in plastid biogenesis. However, the import machinery of cytosolic precursor proteins to plastids relies on the Toc and Tic (translocons on the outer envelope and inner envelope membrane of chloroplasts) complexes. Toc159 protein was identified in pea (Pisum sativum) as a major receptor for the precursor proteins. In Arabidopsis thaliana, four psToc159 homologs are identified, termed atToc159, atToc132, atToc120 and atToc90. The expression of these protein-encoding genes has to be properly regulated, because their gene products must be correctly integrated to appropriate apparatus to perform their functions.ResultsIn order to elucidate the regulatory mechanisms of atTOC159 homologous gene expression, transgenes containing various lengths of the upstream regulatory sequences of atTOC159/atTOC132/atTOC120/atTOC90 and GUS coding sequence were transferred to wild type Arabidopsis. In accordance with the analysis of GUS activity in these transgenic plants at various developmental stages, these homologous genes had distinct expression patterns. AtTOC159 and atTOC90 are preferentially expressed in above-ground tissues, such as cotyledons and leaves. In mature roots, atTOC159 and atTOC132 are expressed at higher levels, while atTOC120 and atTOC90 are expressed at the basal level. All four genes have increased expression level during flower and fruit development, particularly a remarkably high expression level of atTOC159 in later stage of fruit development. Furthermore, leader intron in the 5′ UTR induces the expression level of atTOC159 members in a tissue-specific manner. This is able to up-regulate the atTOC120 expression in roots/leaves/flowers, and the atTOC90 expression in cotyledons/leaves/anthers.ConclusionsThe differential expression of atTOC159 gene members is essential during plastid development, because proper atToc159 isoforms are required to import distinct proteins to the plastids of different tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-40) contains supplementary material, which is available to authorized users.

Simple and Rapid Molecular Techniques for Identification of Amylose Levels in Rice Varieties

The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)10, (CT)11, (CT)16, (CT)17, and (CT)18 were identified. Of these, (CT)11 and (CT)17 were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)10 and (CT)11, while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5′-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.

Functional interconnections of Arabidopsis exon junction complex proteins and genes at multiple steps of gene expression

The exon junction complex (EJC) is deposited on mRNA after splicing and participates in several aspects of RNA metabolism, from intracellular transport to translation. In this work, the functional and molecular interactions of Arabidopsis homologues of Mago, Y14, and PYM, three EJC components that participate in intron-mediated enhancement of gene expression in animals, have been analysed. AtMago, AtY14, and AtPYM are encoded by single genes that show similar expression patterns and contain common regulatory elements, known as site II, that are required for expression. AtPYM and AtY14 are phosphorylated by plant extracts and this modification regulates complex formation between both proteins. In addition, overexpression of AtMago and AtY14 in plants produces an increase in AtPYM protein levels, while overexpression of AtPYM results in increased formation of a complex that contains the three proteins. The effect of AtMago and AtY14 on AtPYM expression is most likely to be due to intron-mediated enhacement of AtPYM expression, since the AtPYM gene contains a leader intron that is required for expression. Indeed, transient transformation asssays indicated that the three proteins are able to increase expression from reporter constructs that contain leader introns required for the expression of different genes. The results indicate that the plant homologues of Mago, Y14, and PYM are closely interconnected, not only through their function as EJC components but also at different steps of their own gene expression mechanisms, probably reflecting the importance of their interaction for the correct expression of plant genes.

Polymorphism in the GBSS gene affects amylose content in US and European rice germplasm

The present study was conducted to investigate the relationship between waxy allelic forms and amylose in European and US rice germplasm. These allelic forms were defined according to the single-nucleotide polymorphisms (SNP) found in the leader intron 5′ splice site (G → T), exon 6 (A → C) and exon 10 (C → T). The combination of these three SNPs accounted for 89.2% of the variation in apparent amylose content in a pedigree of 85 US rice varieties and 93.8% of the variation among 279 accessions in a European germplasm collection. The allelic forms TAC and TCC were found in low amylose varieties. All varieties with intermediate levels of apparent amylose had the GCC allele. High levels of apparent amylose varieties had either the GAT and GAC allele. The sequence AG TTATA in the intron 1 distinguished the low amylose varieties from the other classes regardless of any other base changes. Intermediate amylose varieties can be distinguished from those with high apparent amylose by changes in either exon 6 or exon 10. However the simplest interpretation of the data is that the tyrosine/serine change in exon 6 is responsible for the lower levels of Granule bound starch synthase (GBSS) protein and thus lower levels of amylose in intermediate vs. high amylose verities.

Opposing developmental functions of Agrocybe aegerita galectin (AAL) during mycelia differentiation

Mycelia of basidiomycetes differentiating into fruiting body is a controlled developmental process, however the underlying molecular mechanism remains unknown. In previous work, a novel fungal Agrocybe aegerita galectin (AAL) was isolated from A. aegerita in our laboratory. AAL was shown to promote mycelial differentiation in A. aegerita and Auricularia polytricha, indicating that AAL might function as a conserved fruiting initiator during basidiomycete mycelia development. In the current work, we investigate the role of AAL in mycelia differentiation and fruiting body formation. First, the expression and localization of AAL in mycelia, primordium and fruiting body were assessed by Western blotting and immunohistochemistry. AAL was found to be ubiquitously expressed in the primordium and fruiting body but not in the mycelia. AAL facilitated mycelia congregation and promoted fruiting body production when AAL was applied on mycelia. At the same time, when AAL was spread on potato dextrose agar (PDA) medium prior to mycelia inoculation, mycelia exhibited slowed growth rates, resulting in mycelia cords formation and inhibition of fruiting body formation. The 5′ regulatory sequence of aal was cloned by ‘genome walking’. Here, we show that aal lack introns in the coding region and the upstream 740 bp sequence was characterized by the existence of core promoter elements, which included: two CCAAT boxes (−535/−280), a GC box (−145), a TATA box (−30) and a fungal leader intron within the 5′ UTR. The identification of regulatory expression elements may provide an explanation to the stage-specific and high-level expression of aal during fruiting development.

The leader intron of AtMHX can elicit, in the absence of splicing, low-level intron-mediated enhancement that depends on the internal intron sequence.

BackgroundIntrons stimulate gene expression in a wide range of organisms by increasing the levels of mature mRNA, without affecting mRNA stability. Although introns sometimes function as transcriptional enhancers, they usually stimulate expression by a process termed intron-mediated enhancement (IME). The mechanism of IME is largely unknown. While splicing per se is not sufficient for IME, as evident from the fact that not all introns increase expression, it is not clear yet whether splicing of the enhancing introns is essential for enhancement. The leader intron (LI) of the Arabidopsis AtMHX gene was previously shown to substantially increase the expression of the AtMHX promoter. Here we investigated whether this LI acts as a transcriptional enhancer and whether its splicing is essential for IME.ResultsExpression in transformed Arabidopsis plants of an AtMHX::GUS construct from which the LI was eliminated was similar to a construct that included only the minimal promoter fused to GUS. Yet, almost no expression was seen in constructs that included the LI in addition to the minimal promoter or the LI inserted in various locations in the promoter. While the LI enhanced 272-fold the expression of the weak AtMHX promoter, only a 3-fold enhancement was observed for the strong CaMV 35S promoter. In the context of the AtMHX promoter, an unspliceable version of the LI that had mutated 5' and 3' splice sites mediated a low-level (5-fold) enhancement. Eliminating the internal 320 nt of the 416 nt unspliceable intron resulted in loss of ability to mediate low-level enhancement.ConclusionsAlthough AtMHX promoter shows almost no expression in the absence of its LI, this intron does not act as a transcriptional enhancer and is unable to support expression in the absence of the enhancer elements of the promoter. It is also shown that the same intron can have very different contributions to expression of different promoters. Our results also demonstrate that while splicing is essential for substantial IME, in the absence of splicing low-level enhancement can be obtained. Notably, it is shown that the internal intron sequence plays a significant role in mediating the low-level enhancement of unspliced introns.

Ubiquitous aspartic proteinase as an actor in the stress response in buckwheat

The aspartic protease (FeAP9) gene from buckwheat resembles the exon-intron structure characteristic for typical aspartic proteinases, including the presence of the leader intron in the 5'-UTR. RT PCR experiments and gel protein blot analysis indicated that FeAP9 was present in all analyzed organs: developing seeds, seedlings, flowers, leaves, roots and stems. Using Real-time PCR, we found that FeAP9 expression is upregulated in buckwheat leaves under the influence of different abiotic stresses, including dark, drought and UV-B light, as well as wounding and salicylic acid.

Comparative expression and transcript initiation of three peach dehydrin genes

Dehydrin genes encode proteins with demonstrated cryoprotective and antifreeze activity, and they respond to a variety of abiotic stress conditions that have dehydration as a common component. Two dehydrins from peach (Prunus persica L. [Batsch.]) have been previously characterized; here, we describe the characterization of a third dehydrin from peach bark, PpDhn3, isolated by its response to low temperature. The expression of all three dehydrin genes was profiled by semi-quantitative reverse transcription PCR, and transcript initiation was mapped for all three genes using the RNA ligase-mediated 5' rapid amplification of cDNA ends technique. PpDhn3 transcripts from bark collected in December or July, as well as transcripts from developing fruit, initiated at a single site. Although most of the PpDhn1 transcripts initiated at a similar position, those from young fruit initiated much further upstream of the consensus TATA box. Bark and fruit transcripts encoding PpDhn2 initiated ca. 30 bases downstream of a consensus TATA box; however, transcripts from ripe fruit initiated further upstream. Ripe fruit transcripts of PpDhn2 contain a 5' leader intron which is predicted to add some 34 amino acids to the N-terminal methionine of the cognate protein when properly processed. Secondary structure prediction of sequences surrounding the TATA box suggests that conformational transitions associated with decreasing temperature contribute to the regulation of expression of the cold-responsive dehydrin genes. Taken together these results reveal new, unexpected levels of gene regulation contributing to the overall expression pattern of peach dehydrins.

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 beta-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant beta-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 alpha-tubulin and two rice beta-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.