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Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH-psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra- and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH-psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species.

Angiotensin II (Ang II) type 1 receptor (AGTR1) is a G-protein-coupled receptor (GPCR). Its natural ligand, Ang II, is an important effector molecule controlling blood pressure and volume in the cardiovascular system, and is consequently involved in various diseases such as hypertension and heart failure. Thus, the signaling mediator, AGTR1, is a significant molecular target in medicinal and therapeutic fields. Yeast is a useful organism for sensing GPCR signaling because it provides a simplified version of the complicated machinery used by mammalian cells for signal transduction. Although yeast cells can successfully transmit a signal through a variety of human GPCRs expressed in the cell membrane, there have been no reports of the functional activation of AGTR1-mediated signaling in yeast cells. In the present study, we introduced a single mutation into human AGTR1 and used yeast-human chimeric Gα to exert the functional activation of AGTR1 in yeast cells. The engineered yeast cells expressing AGTR1 mutated at Asn295 and the chimeric Gα successfully transmitted the signal inside the yeast cells in response to Ang II peptide and its analogs (Ang III and Ang IV peptides) added to the assay medium. Further, we demonstrated that the autocrine Ang II peptide and its analog, produced and secreted by the engineered yeast cells, could by themselves promote AGTR1-mediated signaling. This means that screening for agonistic peptides with various sequences from a self-produced genetic library would be a viable strategy. Thus, the constructed yeast biosensor, integrating an Asn295-mutated AGTR1 receptor, will be valuable in the design of drugs to treat AGTR1-related diseases.

c-Myc is one of the most frequently deregulated oncogenes in human cancers, and recent studies showed that even brief inactivation of Myc can be sufficient to induce tumor regression or loss. Consequently, inactivation of Myc provides a novel therapeutic opportunity and challenge, as the dimerization of Myc with Max is crucial for its function. We applied two strategies to specifically target this coiled coil mediated interaction with interfering peptides: a dominant-negative human Max sequence (Max) and a peptide selected from a genetic library (Mip). Both peptides form coiled coils and were fused to an acidic extension interacting with the basic DNA-binding region of human Myc. The genetic library was obtained by semi-rational design randomizing residues important for interaction, and selection was carried out using a protein-fragment complementation assay. The peptides Max and Mip easily outcompeted the human Myc:Max interaction and successfully interfered with the DNA binding of the complex. Both interfering peptides exhibited higher Tm (ΔTm = 13 and 15 °C) upon interaction with Myc compared to wt Max. The inhibitory effect of the two interfering peptides on human Myc:Max activity makes them promising molecules for analytical and therapeutic Myc-directed research. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.

Anyone who has ever spent time in a biochemistry research laboratory can tell you the most problematic reagent in protein science is the protein. Cloning, expression, inclusion bodies, purification, and solubility are just a few of the many pitfalls awaiting the bench scientist on the journey from brilliant idea to published manuscript. Indeed, the value of purified and stable protein became clear (and monetarily quantified) to me during a heavy metal co-crystallization experiment. A back-of-the-envelope-calculation revealed that my protein was worth four orders of magnitude more than the gold I was worried about wasting! Needless to say, obtaining the necessary quantities of protein is often the rate-limiting step in biochemical experimentation. Thankfully, there are many new expression technologies being developed to overcome this barrier. Perhaps most intriguing are cell-free techniques (or in vitro expression), in which the cell is dispensed with altogether, and the protein expression reaction is carried out using reconstituted translation machinery free of the constraints of cellular viability. Cell-free protein synthesis: Methods and protocols is a series of contributions that give an overview of the field including background and history, an in-depth look at the current protocols, and examples of recent successful applications. The book is edited by Alexander Spirin and James Swartz, two of the foremost leaders in the field. Chapter 1, written by the editors, is an introduction. The cell-free reaction consists of a diverse array of biological and chemical components and this chapter is a must-read for anyone new to this science. It describes the evolution of the physical construction of the reaction, the major organismal “platforms” for preparing extracts, and the basis of inclusion for other chemical constituents, including techniques for extending the reaction lifetime using a nucleotide regeneration scheme. Following the introduction, the book is roughly divided in two parts: cell-free reaction development/optimization and the application of cell-free techniques. Chapter 2 is an interesting look at the bottom-up approach to cell-free biology, that is, a protein translation process in which all reaction components (ribosomes, elongation factors, initiation factors, etc.) are purified independently and then reconstituted. This system is an explicitly defined reaction mixture and offers the unique opportunity to study translation (particularly chaperone function) free of the cellular milieu. Chapter 3 describes a system for expression from PCR templates. Such a system could be adapted, in a high-throughput manner, to the functional characterization of entire proteomes. Those looking to bring cell-free techniques into their own laboratory setting may want to pay particular attention to Chapters 5 and 7. These chapters, which focus on bacterial and eukaryotic extracts, respectively, offer up the most practical information in the volume. This includes extract preparation, reaction setup, dialysis, and robotic automation. Much of this practical know-how is not available in the published literature. The second half of the book focuses more on applications. One strength of cell-free expression is the ability to easily incorporate labeled amino acids, and not surprisingly many of the successes have come in structural biology. Perhaps most impressive is the workflow presented by Kigawa et al. in Chapter 6. Here, a cell-free based high-throughput pipeline is outlined that has led to the NMR determination of nearly 1300 structures. Another achievement has been the expression of integral membrane proteins. Integral membrane protein characterization has significantly lagged behind soluble proteins, and Chapters 8 and 9 show that cell-free techniques can be used for the expression of these challenging targets, including even G-protein-coupled receptors. A discussion of the effect of detergents and lipids on the reaction mixture and subsequent protein functionality is also useful. Chapters 10 and 11 focus on using cell-free expression to study “clonal” populations of proteins. In such a setup, single DNA messages can be amplified, transcribed, and translated independently in a microplate or gel and the resulting protein assayed. This holds particular promise in the field of protein evolution and engineering. A genetic library of high-sequence diversity can be used to express many isoforms of a protein, which can in turn be assayed, and the desirable sequences recovered. Finally, the book closes with a look at engineering large-scale (1L+) reactions for industrial production, particularly of therapeutics. Nearly 50 years ago, in its infancy, cell-free expression was harnessed by Nirenberg and colleagues to unravel the genetic code. Spirin and Swartz have assembled here the evolution of this powerful technique, its state-of-the-art, and future applications. The text is clear, readable, and of sufficient detail that protocols can easily be executed by the reader. Cell-free protein synthesis is wholeheartedly recommended for anyone in the field or interested in learning more about protein expression. DAVID F. SAVAGE Department of Systems Biology Harvard Medical School Boston, MA 02115, USA

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.