Resilient Modulus Characterization of Alaskan Granular Base Materials

INE/AUC 11.0

Quikgene: A gene synthesis method integrated with ligation-free cloning

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de nova synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics. (C) 2011 Elsevier Inc. All rights reserved.National Natural Science Foundation of China[30840027, 90919036]; Project 985; Fujian Science Advancing Program[2009J1010]; Project 111[B06016

Dimeric structure of p300/CBP associated factor

National Science Foundation of China [31170685, 90919036, 30840027]; Project 985 [0660ZK1022]; Program 111 [B06016]Background: p300/CBP associating factor (PCAF, also known as KAT2B for lysine acetyltransferase 2B) is a catalytic subunit of megadalton metazoan complex ATAC (Ada-Two-A containing complex) for acetylation of histones. However, relatively little is known about the regulation of the enzymatic activity of PCAF. Results: Here we present two dimeric structures of the PCAF acetyltransferase (HAT) domain. These dimerizations are mediated by either four-helical hydrophobic interactions or a beta-sheet extension. Our chemical cross-linking experiments in combined with site-directed mutagenesis demonstrated that the PCAF HAT domain mainly forms a dimer in solution through one of the observed interfaces. The results of maltose binding protein (MBP)-pulldown, co-immunoprecipitation and multiangle static light scattering experiments further indicated that PCAF dimeric state is detectable and may possibly exist in vivo. Conclusions: Taken together, our structural and biochemical studies indicate that PCAF appears to be a dimer in its functional ATAC complex