Evolution of the tandem repeats in thymidylate synthase enhancer region (TSER) in primates

The upstream regulatory region of the human thymidylate synthase gene (thymidylate synthase enhancer region, TSER) is length polymorphic, attributable to variable numbers of tandemly repeated copies of a 28-bp fragment. It has been found that TSER length

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

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.

Genomic organization, nucleotide sequence analysis of the core histone genes cluster in Chlamys farreri and molecular evolution assessment of the H2A and H2B

This work represents the nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri. The tandemly repeated unit of 5671 bp containing a copy of the four core histone genes H4, H2B, H2A and H3 was amplified and identified by the techniques of homology cloning and genomic DNA walking. All the histone genes in the cluster had the structures in their 3' flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5' region, the transcription initiation sites with a conserved sequence of 5'-PyATTCPu-3' known as the CAP site were present in all genes except to H2B, generally 37-45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5'-GATCC-3' element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5'-GGATCGAAACGTTC-3', which was similar to the consensus sequence of 5'-GGAATAAACGTATTC-3' corresponding to the H2B-specific promoter element. The presence of enhancer sequences (5'-TGATATATG-3') was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes. There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in molluscs.

Characterization and optimization of AuNPs labeled by Raman reporters on glass based on silver enhancement

In this report, gold nanoparticles (AuNPs) labeled by Raman reporters (AuNPs-R6G) were assembled on glass and used as the seeds to in situ grow silver-coated nanostructures based on silver enhancer solution, forming the nanostructures of AuNPs-R6G@Ag, which were characterized by scanning electron microscopy (SEM) and UV-visible spectroscopy. More importantly, the obtained silver-coated nanostructures can be used as a surface enhancement Raman scattering (SERS) substrate. The different SERS activities can be controlled by the silver deposition time and assembly time of AuNPs-R6G on glass. The results indicate that the maximum SERS activity could be obtained on AuNPs-R6G when these nanostructures were assembled on glass for 2 h with silver deposition for 2 min.

The effect of nocodazole on the transfection efficiency of lipid-bilayer coated gold nanoparticles

Nonviral vectors are safer than viral systems for gene therapy applications. However, the limited efficacy always prevents their being widely used in clinical practice. Aside from searching new gene nonviral vectors, many researchers focus on finding out new substances to improve the transfection efficiency of existent vectors. In this work, we found a transfection enhancer, nocodazole (NCZ), for dimethyldioctadecylammonium (DODAB, a cationic lipid) bilayer coated gold nanoparticles (AuNPs) mediated gene delivery. It was found that NCZ produces 3-fold transfection enhancement to HEK 293T cells assessed by flow cytometry (FCM). The result was further confirmed by luciferase assay, in which NCZ induced more than 5 times improvement in transfection efficiency after 48 h of transfection. The results from the inductively coupled plasma mass spectrometry (ICP-MS) and FCM showed that NCZ did not affect the internalization of DODAB-AuNPs/DNA complexes. The trafficking of the complexes by transmission electron microscopy (TEM) indicated that the interrupted transportation of the complexes to the lysosomes contributed greatly to the transfection enhancement.

Interaction of low-molecular-weight chitosan with mimic membrane studied by electrochemical methods and surface plasmon resonance

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by-fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed,to be in a concentration-dependent manner.