萝卜-芥蓝异源四倍体植物SR30基因转录本的选择性剪接分析

为了解异源多倍体形成后,其剪接因子基因SR30在各组织器官间的表达量以及选择性剪接模式与亲本的差异,选取萝卜-芥蓝异源四倍体(Raphanobrassica)及其亲本萝卜(Raphanus sativus)、芥蓝(Brassica oleracea var.alboglabra)为材料,运用RACE-PCR方法克隆到全长的编码序列(CDS)和3非编码区(3 UTR),运用q RT-PCR和半定量RT-PCR检测其在各组织器官中的表达量和各转录本表达量间的差异。结果表明,四倍体中萝卜同源的Rs SR30基因有5种转录本,芥蓝同源的Bo SR30基因有4种转录本。同时,SR30在3物种中的表达具有组织器官的差异,且在四倍体中的总体表达量显著低于亲本。根据克隆到的转录本,预测Rs SR30编码3种蛋白,Bo SR30编码2种,不同蛋白异构体的区别体现在C末端的丝氨酸-精氨酸富集(RS)结构域。因此,萝卜-芥蓝异源多倍体形成后,SR30基因在表达量和转录本选择性剪接方面都发生了改变。     

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene ( KLC1 ) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.     

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Activities of superoxide dismutase (SOD) isoforms during growth of <Emphasis Type="Italic">Scenedesmus</Emphasis> (chlorophyta) species and strains grown in batch-cultures

Growth of Scenedesmus species and strains, grown for 28 days in mineral BBM medium in batch-cultures, displayed sigmoidal kinetics that comprised a lag, exponential and declining growth phases. Total SOD activity in these autotrophically cultured organisms, which oscillated within 0.6 – 1.4 Umg protein⁻¹, was rather species-specific and only to some extent depended on the growth phase. Contrary, three S. obliquus strains: wild type 276-6, mutant with blocked PS I (strain 56.80) and mutant with blocked PS II (strain 57.80), cultured for 7 days on BBM medium supplemented with bacto-tryptone and yeast extract (BBM+) turned out to be time-dependent and to have several times higher total SOD activity than one obtained for Scenedesmus grown autotrophically. Regardless of the media composition, the phase of growth and studied organism, dominant isoforms of total SOD were together determined Fe- and Mn-SOD. Profiles of SOD isoforms, obtained after PAGE analysis of all autotrophically and exponentially growing organisms, revealed that one Mn-SOD and one Cu/Zn-SOD bands located on gels at the same position whereas location of three bands of Fe-SOD depended on the strain. This suggests the presence of two different groups of Fe-SODs in analyzed organisms. Identical SOD profiles found in two S. armatus strains (276-4a and 276-4d) and S. subspicatus correspond well with their taxonomic position. The SOD profile of S. armatus B1-76 distinctly differed from two other S. armatus strains but was identical to S. microspinal B1-76 and S. quadricauda G-15 despite the fact that there were significant growth rate differences between these three species. SODs profiles of S. acutus 437 and S. obliguus 453 were species-specific. In S. obliquus strains cultured on BBM+ medium, there are four SOD bands: one slightly visible band of Mn-SOD, two intensive bands of Fe-SOD and one band of Cu/Zn-SOD. The above finding suggests that antioxidant response of algae kept in batch-cultures differs according to medium composition and the SOD activity mainly restricted to chloroplasts.     

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H₂O₂-and o-dianisidine/H₂O₂-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.This work was supported in part by the scientific Research Foundaation (OTKA F 5082), the foundation for Hungarian science, a british council scolership to A.L.A and the U.K. Agricultural and food Reaserch council.     

A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch

Smooth muscle myosin phosphatasedephosphorylates the regulatory myosin light chain and thus mediatessmooth muscle relaxation. The activity of this myosin phosphatase isdependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle sectionof chicken and rat MYPT1 genes revealed that each gene gave rise toisoforms by cassette-type alternative splicing of exons. In chicken, a123-nucleotide exon was included or excluded from the mature mRNA,whereas in rat two exons immediately downstream were alternative. MYPT1isoforms lacking the alternative exon were only detected in maturechicken smooth muscle tissues that display phasic contractileproperties, but the isoform ratios were variable. The patterns ofexpression of rat MYPT1 mRNA isoforms were more complex, with threemajor and two minor isoforms present in all smooth muscle tissues atvarying stoichiometries. Isoform switching was identified in thedeveloping chicken gizzard, in which the exon-skipped isoform replacedthe exon-included isoform around the time of hatching. This isoformswitch occurred after transitions in myosin heavy chain and myosinlight chain (MLC₁₇) isoforms and correlated with aseveralfold increase in the rate of relaxation. The developmentalswitch of MYPT1 isoforms is a good model for determining the mechanismsand significance of alternative splicing in smooth muscle.

     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Antioxidant Enzyme Isoforms on Gels in Two Poplar Clones Differing in Sensitivity After Exposure to Ozone

The effect of acute ozone (O₃) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O₃-sensitivity was analysed. Untreated leaves of both the O₃-sensitive (O₃-S) clone Eridano of Populus deltoides×P. maximowiczii and the O₃-resistant (O₃-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (R_f) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O₃-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O₃-R plants seven POD isoforms (R_f= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (R_f= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O₃-S plants were resolved. The electrophoretic pattern of POD in O₃-S leaves was greatly modified by acute O₃-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (R_f= 0.13) in YL. Additionally, O₃-exposure induced the appearance of two APX isoforms in YL (R_f= 0.66 and 0.70), and one isoform in ML (R_f= 0.70) of the O₃-S clone. By contrast, the activity of the three APX isoformes (R_f= 0.64, 0.70 and 0.76) detected in O₃-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O₃ stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O₃-sensitivity.     

ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform

Background Tissue-specific alternative pre-mRNA splicing is a widely used mechanism for gene regulation and the generation of different protein isoforms, but relatively little is known about the factors and mechanisms that mediate this process. Tissue-specific RNA-binding proteins could mediate alternative pre-mRNA splicing. In Drosophila melanogaster, the RNA-binding protein encoded by the elav (embryonic lethal abnormal visual system) gene is a candidate for such a role. The ELAV protein is expressed exclusively in neurons, and is important for the formation and maintenance of the nervous system.Results In this study, photoreceptor neurons genetically depleted of ELAV, and elav-null central nervous system neurons, were analyzed immunocytochemically for the expression of neural proteins. In both situations, the lack of ELAV corresponded with a decrease in the immunohistochemical signal of the neural-specific isoform of Neuroglian, which is generated by alternative splicing. Furthermore, when ELAV was expressed ectopically in cells that normally express only the non-neural isoform of Neuroglian, we observed the generation of the neural isoform of Neuroglian.ConclusionsDrosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Prompt response of superoxide dismutase and peroxidase to dehydration and rehydration of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The relic endemic nature of Haberlea rhodopensis, which grows in Balkan Peninsula, in combination with its high vegetative desiccation-tolerance, makes this species a good model to study mechanisms behind plant adaptation to severe drought stress. The aim of this study was to evaluate the antioxidant protection provided by Superoxide dismutase (SOD) and Peroxidase (PO) in H. rhodopensis after exposure to and recovery from dehydration at different developmental stages. During dehydration the electrolyte leakage from leaf tissue increased more significantly in post-flowering plants than in flowering plants, while upon subsequent rehydration this parameter showed a very fast decrease to the basic value of fresh leaves and did not depend on developmental stage. Like other higher plant species, SOD and PO demonstrated in H. rhodopensis an ability to adjust their activity very promptly to changing water supply. In addition, the leaves of this resurrection species retained significant activities of SOD and PO even in air-dried state, considered as the most severe form of water stress. The enhanced activity of antioxidant enzymes may either enable the scavenging of the active oxygen species produced at very severe water deficit, and/or carry a potential for resurrection on subsequent rehydration. Upon stress treatment total activities of both enzymes were higher in flowering than post-flowering plants which reveals that developmental stage might be a factor affecting plant stress tolerance. This work identified for the first time SOD isoforms of H. rhodopensis. Native PAGE showed at least six multiple isoforms in the protein extract from leaf tissue of flowering plants, and the differential visualization revealed that four of them were Cu, Zn-SOD isoforms, one was Mn-SOD and one Fe-SOD. These findings provide a good starting point for future study of the SOD gene family of this rare resurrection plant at the molecular level.