The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein

MEP is a member of thioester-containing protein (TEP) family found in Zhikong scallop Chlamys farreri and is involved in innate immunity against invading microbes. In the present study, the genomic DNA of CfTEP was cloned and characterized. The genomic DNA sequence of CfTEP consisted of 40 exons and 39 introns spanning 35 kb with all exon-intron junction sequences agreeing with the GT/AG consensus. The genomic organization of CfTEP was similar to human and mouse 0 rather than ciona C3-1 and Drosophila dTEP2. By RT-PCR technique, seven different cDNA variants of CfTEP (designated as CfTEP-A-CfTEP-G) were cloned from scallop gonad. CfTEP-A-CfTEP-F were produced by alternative splicing of six mutually exclusive exons (exons 19-24), respectively, which encoded the highly variable central region. While in CfTEP-G, the deletion of all the six exons introduced a new translation stop site and might trigger nonsense mediated decay (NMD). The mRNA expression and the proportion of the seven CfTEP variant transcripts were examined in the gonad of scallops after bacterial challenge. The fragments containing the highly variable central region of UTEP were amplified by RT-PCR and a 100 positive clones were sequenced randomly. The expression profiles of the seven MEP variants were different and displayed the sex and bacteria dependent manner. In the blank, sea water and Listonella anguillarum challenged subgroups of male scallops, all the transcripts detected were CfTEP-G isoform. In the Micrococcus luteus challenged subgroup, the isoforms expressed and their proportions were CfTEP-F (54%), CfTEP-B (23%), CfTEP-A (10%), CfTEP-C (7%) and CfTEP-E (6%). However, in the gonad of female scallops, only CfTEP-A were found in blank and sea water challenged subgroups. After L anguillarum or M. luteus challenge, four and five isoforms were detected, respectively, with CfTEP-F isoform being the most one in the both subgroups. These results suggested that the evolution of TEP genes was very complex, and that the diverse CfTEP transcripts generated by alternative splicing played an important role as pattern recognition receptors in the innate immune defense of scallops. (C) 2009 Elsevier Ltd. All rights reserved.

Alternative splicing of the FMR1 gene in human tissues of fetal and adult

To study the time- and tissue-specificity of alternative splicing of the FMR1 gene, we analyzed the alternative splicing pattern of the FMR1 gene in human tissues from adult and fetus using RT-PCR coupled with capillary electrophoresis. Seven alternative splicing variants of FMR1 were found in adult liver and lung. The major three alternative splicing variants of the FMR1 gene in all analyzed fetal tissues were same, though the number of minor isoforms and the relative abundance of major isoforms were different. The major difference of the alternative splicing pat tem between adult and fetus was in exon 12 and 17. The results suggest that the alternative splicing pattern of the FMR1 gene is non-tissue-specific in the same developmental stage and a developmental switch may be present.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

Functional characterization of the human-specific (type II) form of kallikrein 8, a gene involved in learning and memory

Kallikrein 8 (KLK8) is a serine protease functioning in the central nervous system, and essential in many aspects of neuronal activities. Sequence comparison and gene expression analysis among diverse primate species identified a human-specific splice for

Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification

Antimicrobial peptides secreted by the skin of many amphibians play an important role in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA sequences, each of which encodes a precursor peptide that can give rise to two kinds of antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating the diversity of maximins and maximin Hs. The gene trees based on different domain regions revealed that domain shuffling or gene conversion among these genes might have happened frequently.

Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in similar to 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

Identification and characterization of a novel splice variant of gonadotropin alpha subunit in the common carp Cyprinus carpio

In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

Molecular cloning of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

Precipitation of nanocrystals in glasses by electron irradiation: An alternative path to form glass ceramics?

This letter demonstrates an alternative method to form gallium silicate glass ceramics using high-energy electron irradiation. Compared with glass ceramics obtained from the conventional thermal treatment method, the distribution and crystal sizes of the precipitated Ga2O3 nanoparticles are the same. An advantage of this method is that the spatial distribution of the precipitated nanoparticles can be easily controlled. However, optically active dopants Ni2+ ions do not participate in the precipitation during electron irradiation. (c) 2007 American Institute of Physics.

Transparent colloid containing upconverting nanocrystals: An alternative medium for three-dimensional volumetric display

We report an alternative medium of transparent upconverting colloid containing lanthanide ion doped NaYF4 nanocrystals for three-dimensional (3D) volumetric display. The colloids exhibit tunable upconversion luminescence with a wide spectrum of colors by adjusting the doping concentrations of the nanocrystals and the compositions of the colloids. Our preliminary experimental result indicates that an upconverting colloid-based 3D volumetric display using a convergent, near infrared laser beam to induce a localized luminescent spot near the focus is technically feasible. Therefore arbitrary 3D objects can be created inside the upconverting colloid by use of computer controlled 3D scanning systems. (C) 2008 Optical Society of America

Secondary structures of mRNAs and splicing of introns in eukaryotes.

After analyzing the secondary structures of 68 exon-intron-exon and the corresponding exon-exon sequence segments, it is found that about 90% of 5' and 3' terminal bases G (splicing sites) of introns are situated in the loops of secondary structures or at the ends of stems near the loops, and most of "G" s in loops are closed to the ends of loops. Approximately 92% of the connecting sites of the adjoining exons also show the similar features. About 82% of the branch point "A" s are situated in loops or at the ends of stems near the loops. Splicing sites and branch points approach each other in space because of the folding.

Alternative cold response modes in Chlorella (Chlorophyta, Trebouxiophyceae) from Antarctica

Chlorella was known to show enhanced antifreeze capability after cold hardening. We isolated Chlorella strains NJ-7 and NJ-18, which display alternative cold response modes from rock surfaces in Antarctica. On the basis of 18S ribosomal (rRNA) sequences, NJ-7 is an Antarctic type of Chlorella vulgaris; NJ-18 is also a 'true' Chlorella species but differs from any previously reported species in structure. NJ-7 partially retained the enhancing effects of low temperature cultivation on freeze tolerance, which correlates with an increase of C18:3-fatty acid content and up-regulation of two antifreeze protein genes. NJ-18, however, showed stable freeze tolerance regardless of the precultivation temperature. We propose that cold response modes vary widely in Chlorella and that the adaptation of C. vulgaris to Antarctica may serve as a model system for the evolution of antifreeze mechanisms in a single species of photosynthetic microorganism.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.