Jun Hu

A DNA-Origami Chip Platform for Label-Free SNP Genotyping Using Toehold-Mediated Strand Displacement

[Zhang, Z; Feng, GY; He, L; Li, C; Fan, CH] Shanghai Jiao Tong Univ, Key Lab Genet Dev & Neuropsychiat Disorders, Minist Educ, BioX Ctr, Shanghai 200030, Peoples R China; [Zeng, DD; Hu, J; Fan, CH] Chinese Acad Sci, Phys Biol Lab, Shanghai Inst Appl Phys, Shanghai 201800, Peoples R China; [Zeng, DD; Ma, HW] Chinese Acad Sci, Suzhou Inst Nanotech & Nanobion, Suzhou 215125, Peoples R China; [Zhang, Z] Aarhus Univ, Ctr DNA Nanotechnol CDNA, DK-8000 Aarhus C, Denmark

Modulation of DNA Polymerases with Gold Nanoparticles and their Applications in Hot-Start PCR

A new gold-nanoparticle (AuNP)-based strategy to dynamically modulate the activity of DNA polymerases and realize a hot-start (HS)-like effect in the polymerase chain reaction (PCR) is reported, which effectively prevents unwanted nonspecific amplification and improves the performance of PCRs. A high-fidelity Pfu DNA polymerase is employed as the model system. Interestingly, AuNPs inactivate the polymerase activity of Pfu at low temperature, thus resembling an antibody-based HS PCR. This inhibition effect of AuNPs is demonstrated for the preamplification polymerization activity of the PCR, which largely suppresses nonspecific amplification at temperatures between 30 and 60 degrees C and leads to highly specific and sensitive PCR amplification with Pfu. Significantly, the fidelity of Pfu is not sacrificed in the presence of AuNPs. Therefore, this AuNP-based HS strategy provides a straightforward and potentially versatile approach to realize high-performance PCR amplification.

Identification and functional analysis of the genes encoding Δ6-desaturase from Ribes nigrum†

Gamma-linolenic acid (γ-linolenic acid, GLA; C18:3 Δ6, 9, 12) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Δ6-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Δ6-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Δ6-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Δ6-desaturases that could use both α-linolenic acids (ALA; C18:3 Δ9,12,15) and linoleic acid (LA; C18:2 Δ9,12) precursors in vivo, whereas RnD8A/B were Δ8-sphlingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Δ6-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Δ6,9,12,15) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Stretching and imaging studies of single DNA molecules

DNA molecules were stretched on silanized mica surface with the molecular combing technique, and detected with fluorescence microscopy and atomic force microscopy. Meantime, DNA molecules were stretched with a modified dynamic molecular combing technique and studied with atomic force microscopy. The results indicate that, compared with the dynamic molecular combing technique, the modified dynamic molecular combing technique has advantages of less-sample demand and less contamination to sample; as compared with the molecular combing technique, it has better aligning effect and reproducibility. Combination of this kind of DNA molecular manipulating technique with the single DNA molecule detecting technique by atomic force microscopy and fluorescence microscopy will play an important role in the basic research of molecular dynamics and the application of gene research.

GlnR Negatively Regulates the Transcription of the Alanine Dehydrogenase Encoding Gene ald in Amycolatopsis mediterranei U32 under Nitrogen Limited Conditions via Specific Binding to Its Major Transcription Initiation Site

Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT) and alanine dehydrogenase (AlaDH) in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs) of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II) were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.

Enhanced tolerance to NaCl and LiCl stresses by over-expressing Caragana korshinskii sodium/proton exchanger 1 (CkNHX1) and the hydrophilic C terminus is required for the activity of CkNHX1 in Atsos3-1 mutant and yeast.

Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na(+)/H(+) transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii, a pea shrub with high tolerance to salt, drought, and cold stresses. Transcripts of CkNHX1 were detected predominantly in roots, and were significantly induced by NaCl stress in stems. Transgenic yeast and Arabidopsisthalianasos3-1 (Atsos3-1) mutant over-expressing CkNHX1 and its hydrophilic C terminus-truncated derivative, CkNHX1-ΔC, were generated and subjected to NaCl and LiCl stresses. Expression of CkNHX1 significantly enhanced the resistance to NaCl and LiCl stresses in yeast and Atsos3-1 mutant. Whereas, compared with expression of CkNHX1, the expression of CkNHX1-ΔC had much less effect on NaCl tolerance in Atsos3-1 and LiCl tolerance in yeast and Atsos3-1. All together, these results suggest that the predominant expression of CkNHX1 in roots might contribute to keep C. korshinskii adapting to the high salt condition in this plants living environment; CkNHX1 could recover the phenotype of Atsos3-1 mutant; and the hydrophilic C-terminal region of CkNHX1 should be required for Na(+)/H(+) and Li(+)/H(+) exchanging activity of CkNHX1.

Fabrication of true-color micropatterns by 2D stepwise contraction and adsorption nanolithography (scan)

Fabrication of structures on the micro- and nanometer scales is of great importance for both fundamental research and potential applications. While microlithography methods are relatively established, the production of multi-component micro- and nanostructures with high density still presents difficulties. In this paper, a novel strategy termed as two-dimensional (2D) stepwise contraction and adsorption nanolithography (SCAN) is used to fabricate true-color micropatterns through a series of size-reduction process based on the physical elasticity of elastomer. Faithful multicolor patterns with feature size about 30 times smaller than the initial ones can be fabricated by employing the 2D SCAN. The simplicity and high throughput capability of SCAN make it a competitive alternative to other micro- and nanolithography techniques.

Synchrotron FTIR microspectroscopy reveals early adipogenic differentiation of human mesenchymal stem cells at single-cell level.

Human mesenchymal stem cells (hMSCs) have been used as an ideal inxa0vitro model to study human adipogenesis. However, little knowledge of the early stage differentiation greatly hinders our understanding on the mechanism of the adipogenesis processes. In this study, synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was applied to track the global structural and compositional changes of lipids, proteins and nucleic acids inside individual hMSCs along the time course. The multivariate analysis of the SR-FTIR spectra distinguished the dynamic and significant changes of the lipids and nucleic acid at early differentiation stage. Importantly, changes of lipid structure during early days (Day 1-3) of differentiation might serve as a potential biomarker in identifying the state in early differentiation at single cell level. These results proved that SR-FTIR is a powerful tool to study the stem cell fate determination and early lipogenesis events.