Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism.

The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8-4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent-buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25-37%, and PI isomeric purities of 91-96%.

Comprehensive analysis of sphingosine-1-phosphate receptor mutants during zebrafish embryogenesis.

The lipid mediator sphingosine-1-phosphate (S1P) regulates various physiological and pathological phenomena such as angiogenesis and oncogenesis. Secreted S1P associates with the G-protein-coupled S1P receptors (S1PRs), leading to the activation of downstream signaling molecules. In mammals, five S1prs have been identified and the genetic disruption of a single S1pr1 gene causes vascular defects. In zebrafish, seven s1prs have been isolated. We found that individual s1prs showed unique expression patterns with some overlapping expression domains during early embryogenesis. We generated all s1pr single-mutant zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases and analyzed their phenotypes during early embryogenesis. Zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants showed no developmental defects and grew into adults, whereas zygotic s1pr2 mutant showed embryonic lethality with a cardiac defect, showing quite distinct embryonic phenotypes for individual S1pr mutants between zebrafish and mouse. We further generated maternal-zygotic s1pr1, s1pr3a, s1pr3b, s1pr4, s1pr5a and s1pr5b mutants and found that these maternal-zygotic mutants also showed no obvious developmental defects, presumably suggesting the redundant functions of the S1P receptor-mediated signaling in zebrafish.

Genetically encoded system to track histone modification in vivo.

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1-10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.

Quantitative assay for TALEN activity at endogenous genomic loci.

Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue-white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.

Combination of Clk family kinase and SRp75 modulates alternative splicing of Adenovirus E1A.

SR proteins are non-snRNP splicing factors harbouring a domain rich in Arg-Ser repeats, which are extensively phosphorylated by several kinases. We performed a comparative study of different SR kinases, including SRPK, Clk, PRP4 and DYRK, and found that only Clks efficiently altered 5' splice site selection of Adenovirus E1A. The phosphorylation state of SR proteins was examined using a phospho-SR specific antibody mAb1H4 and a 75 kDa protein was most evidently hyperphosphorylated by Clks. Administration of TG003, a specific inhibitor for the Clk family members, specifically and rapidly induced dephosphorylation of 75 kDa SR protein. Imaging with mRFP-SRp75 in living cells revealed that its nuclear distribution was rapidly altered upon inhibition of the Clk activity by TG003. Co-transfection experiments demonstrated that HA-tagged SRp75 was hyperphosphorylated by Clk family members, but not by other SR kinases. These results indicate that Clks specifically hyperphosphorylate SRp75. Furthermore, SRp75 over-expression promoted the selection of 12S 5' splice site in E1A pre-mRNA, which is stimulated by co-expression of Clks. These results suggest that the specific combination of SR protein and SR kinase plays a distinct role in alternative splicing through dynamic balance of phosphorylation.

A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B.

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A-H2B, we identified protein phosphatase (PP) 2C gamma subtype (PP2Cgamma/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A-H2B. The disruption of PP2Cgamma in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cgamma-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.

Manipulation of alternative splicing by a newly developed inhibitor of Clks.

The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASF-dependent splicing of beta-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.