A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation.

Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.

Disruption of the interaction between TFIIAαß and TFIIA recognition element inhibits RNA polymerase II gene transcription in a promoter context-dependent manner.

General transcription factors and core promoter elements play a pivotal role in RNA polymerase II (Pol II)-mediated transcription initiation. In the previous work, we have defined a TFIIA recognition element (IIARE) that modulates Pol II-directed gene transcription in a promoter context-dependent manner. However, how TFIIA interacts with the IIARE and whether the interaction between TFIIA and the IIARE is involved in the regulation of gene transcription by Pol II are not fully understood. In the present study, we confirm that both K348 and K350 residues in TFIIAαß are required for the interaction between TFIIAαß and the IIARE. Disruption of the interaction between them by gene mutations dampens TFIIAαß binding to the AdML-IIARE promoter and the transcriptional activation of the promoter containing a IIARE in vitro and in vivo. Stable expression of the TFIIAαß mutant containing both K348A and K350A in the cell line with endogenous TFIIAαß silence represses endogenous gene expression by reducing the occupancies of TFIIAαß, TBP, p300, and Pol II at the promoters containing a IIARE. The findings from this study provide a novel insight into the regulatory mechanism of gene transcription mediated by TFIIA and the IIARE.

The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling BRF1 and GTF3C2 expression in human cells.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

An optimized method for neuronal differentiation of embryonic stem cells in vitro.

BACKGROUND: Neural differentiation from embryonic stem cells (ESCs) is an excellent model for elucidating the key mechanisms involved in neurogenesis, and also provides an unlimited source of progenitors for cell-based nerve regeneration. However, the existing protocols such as small molecule substances, 3D matrix, co-culture technique and transgenic method, are complicated and difficult to operate, thus are limited by laboratory conditions. Looking for an easy-to-operate protocol with easily gained material and high induction efficiency has always been a hot issue in neuroscience research. NEW METHODS: This paper established an optimized method for embryonic neurogenesis using a strategy of "combinatorial screening". In our study, the whole process of embryonic neurogenesis was divided into two phases, and the differentiation efficiency of seven experimental protocols in phase I and three protocols in phase II were systematically evaluated in A2lox and 129 ESCs. RESULTS: In phase I differentiation, "2-day embryoid bodies formation + 6-day retinoic acid induction" (Phase I-protocol 3) could effectively induce the differentiation of ESCs into neural precursor cells (NPCs). Furthermore, in phase II, N2B27 medium II (Phase II-protocol 3) could better support the subsequent differentiation from NPCs into neurons. COMPARISON WITH EXISTING METHOD(S): Such a combinational method (phase I-protocol 3 and phase II-protocol 3) can realize embryonic neurogenesis with high efficiency, easy implementation and low-cost, and is suitable for promotion in most laboratories. CONCLUSIONS: Through "combinatorial screening" strategy, we established an optimized method for embryonic neurogenesis in vitro, which is expected to be a powerful tool for neuroscience research.

The 40Ar/39Ar dating of quartz: new insights into the metallogenic chronology of the Jinchang gold deposit and its geological significance.

The Jinchang gold deposit has been extensively studied, but precise dates for its formation are debated. Native gold mainly occurs as inclusions within pyrite and quartz. In this study, we analysed quartz crystals coeval with gold precipitation from two different types of mineralization using the ArgusVI multi-collector noble gas mass spectrometer by the stepwise crushing technique to resolve the timing and genesis of gold mineralization. 40Ar/39Ar dating of quartz samples (J12Q) from breccia ore yields a plateau age of 109.87 ± 0.86 Ma, and an inverse isochron age of 109.87 ± 0.88 Ma. Quartz samples (J18Q) from vein ore yields a slightly younger plateau age of 107.76 ± 0.85 Ma, with an inverse isochron age of 107.76 ± 0.92 Ma. These dates place the ore-forming age of the Jinchang gold deposit at 107~110 Ma, much younger than previously published radiometric ages, suggesting the gold mineralization is spatio-temporally associated with the granite porphyry. The formation of the Jinchang gold deposit is consistent with the regional late Mesozoic porphyry-epithermal gold mineralization event in the Yanbian-Dongning area. Finally, our study shows that 40Ar/39Ar of quartz can be used as a powerful tool to date the formation ages of hydrothermal ore deposits.