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Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel β-barrel, which is open to the solvent at one end. There, four loops connect the β-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.

The engineering of synthetic gene networks has mostly relied on the assembly of few characterized regulatory elements using rational design principles. It is of outmost importance to analyze the scalability and limits of such a design workflow. To analyze the design capabilities of libraries of regulatory elements, we have developed the first automated design approach that combines such elements to search the genotype space associated to a given phenotypic behavior. Herein, we calculated the designability of dynamical functions obtained from circuits assembled with a given genetic library. By designing circuits working as amplitude filters, pulse counters and oscillators, we could infer new mechanisms for such behaviors. We also highlighted the hierarchical design and the optimization of the interface between devices. We dissected the functional diversity of a constrained library and we found that even such libraries can provide a rich variety of behaviors. We also found that intrinsic noise slightly reduces the designability of digital circuits, but it increases the designability of oscillators. Finally, we analyzed the robust design as a strategy to counteract the evolvability and noise in gene expression of the engineered circuits within a cellular background, obtaining mechanisms for robustness through non-linear negative feedback loops, Funding for open access charge: Generalitat Valenciana (BFPI-2007-160); HPC-Europa programme (RII3-CT- 2003-506079); Spanish Ministry of Education and Science (TIN-2006-12860); Structural Funds ERDF;FP6-NEST 043340 (BioModularH2); FP7-ICT-043338 (BACTOCOM); FP7-ICT-265505 (CADMAD); ATIGE Genopole/UEVE (A3405); Fondation pour la Recherche Medicale.

DNA error correcting codes over the edit metric consist of embeddable markers for sequencing projects that are tolerant of sequencing errors. When a genetic library has multiple sources for its sequences, use of embedded markers permit tracking of sequence origin. This study compares different methods for synthesizing DNA error correcting codes. A new code-finding technique called the salmon algorithm is introduced and used to improve the size of best known codes in five difficult cases of the problem, including the most studied case: length six, distance three codes. An updated table of the best known code sizes with 36 improved values, resulting from three different algorithms, is presented. Mathematical background results for the problem from multiple sources are summarized. A discussion of practical details that arise in application, including biological design and decoding, is also given in this study.

A genetic library of tributyltin chloride (TBTC1)-resistant marine bacterium, Alteromonas sp. M-1, was constructed using plasmid vector pUC 19. Three positive clones were obtained from E. coli JM 109 transformed with the plasmids by the method of replica plating to LB medium containing 1 mM TBTC1. These clones could grow in LB liquid medium containing 100 microM TBTC1. Plasmids harbouring genes of Alteromonas sp. M-1 were designated pTBT1, pTBT2 and pTBT3 which contain 1.8 kb Hind III-fragment, 4.8 kb Pst I-fragment and 7.8 kb Pst I-fragment, respectively. Nucleotide sequence of the shortest fragment, 1.8 kb Hind III-fragment was determined, revealing an open reading frame (ORF) was contained in the fragment. The ORF was 324 bp (108 amino acids). The 48.5% of the amino acids encoded was hydrophobic, suggesting that the product relating to TBTC1 resistance might be membrane related protein. Homology search in amino acids alignment indicated that the product has homology with transport proteins.

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.

Made available in DSpace on 2013-08-12T19:03:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Made available in DSpace on 2013-09-30T17:49:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:13:14Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:13:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-01 Diesel oil is a compound derived from petroleum, consisting primarily of hydrocarbons. Poor conditions in transportation and storage of this product can contribute significantly to accidental spills causing serious ecological problems in soil and water and affecting the diversity of the microbial environment. The cloning and sequencing of the 16S rRNA gene is one of the molecular techniques that allows estimation and comparison of the microbial diversity in different environmental samples. The aim of this work was to estimate the diversity of microorganisms from the Bacteria domain in a consortium specialized in diesel oil degradation through partial sequencing of the 16S rRNA gene. After the extraction of DNA metagenomics, the material was amplified by PCR reaction using specific oligonucleotide primers for the 16S rRNA gene. The PCR products were cloned into a pGEM-T-Easy vector (Promega), and Escherichia coli was used as the host cell for recombinant DNAs. The partial clone sequencing was obtained using universal oligonucleotide primers from the vector. The genetic library obtained generated 431 clones. All the sequenced clones presented similarity to phylum Proteobacteria, with Gammaproteobacteria the most present group (49.8 % of the clones), followed by Alphaproteobacteira (44.8 %) and Betaproteobacteria (5.4 %). The Pseudomonas genus was the most abundant in the metagenomic library, followed by the Parvibaculum and the Sphingobium genus, respectively. After partial sequencing of the 16S rRNA, the diversity of the bacterial consortium was estimated using DOTUR software. When comparing these sequences to the database from the National Center for Biotechnology Information (NCBI), a strong correlation was found between the data generated by the software used and the data deposited in NCBI. UNESP São Paulo State Univ, FCAV, Dept Biol Appl Agr & Anim Husb Dev, BR-14884900 Jaboticabal, SP, Brazil Univ São Paulo, BR-05508 São Paulo, Brazil Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Soybean, BR-86001970 Londrina, Parana, Brazil UNESP São Paulo State Univ, FCAV, Dept Biol Appl Agr & Anim Husb Dev, BR-14884900 Jaboticabal, SP, Brazil

Water contamination by nitrate is a wideworld extended phenomena. Biological autotrophic denitrification has a real potential to face this problem and presents less drawbacks than the most extended heterotrophic denitrification. Three bench-scale UASB reactors were operated under autotrophic (R1, H2S as electron donor), mixotrophic (R2, H2S plus p-cresol as electron donors) and heterotrophic (R3, p-cresol as electron donor) conditions using nitrate as terminal electron acceptor. 16S rDNA genetic libraries were built up to compare their microbial biodiversity. Six different bacteria phyla and three archaeal classes were observed. Proteobacteria was the main phyla in all reactors standing out the presence of denitrifiers. Microorganisms similar to Thiobacillus denitrificans and Acidovorax sp. performed the autotrophic denitification. These OTUs were displaced by chemoheterotrophic denitrifiers, especially by Limnobacter-like and Ottowia-like OTUs. Other phyla were Bacteroidetes, Chloroflexi, Firmicutes and Actinobacteria that - as well as Archaea members - were implicated in the degradation of organic matter, as substrate added as coming from endogenous sludge decay under autotrophic conditions. Archaea diversity remained low in all the reactors being Methanosaeta concilii the most abundant one.

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding alpha3beta3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both alpha-helix and beta-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three alpha-helices and three beta-strands as intended by its design. Thus, it would appear that artificially generated alpha3beta3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.

Gene therapy, to delivery of genetic material to a patient for therapeutic benefit, has significant promise for translating basic knowledge of disease mechanism into biomedical treatments. The clinical development of the field has been slowed, however, by the need for improvements in the properties and capabilities of gene delivery vehicles. Vehicles based on viruses offer the potential for efficient gene delivery, but because viruses did not evolve to serve human therapeutic needs, many of their properties require significant improvement, including their safety, efficiency, and capacity for targeted gene delivery. Since viruses are highly complex biological entities, engineering such properties at the molecular level can be challenging. However, there has been significant progress in developing approaches that mimic the mechanisms by which viruses arose in the first place. In particular, library-based selection, the generation of one diverse genetic library and selection for new properties, and directed evolution, based on the multiple rounds of library generation and selection for iterative improvement of function, have strong potential in engineering novel properties into these complex biomolecular assemblies. This review will discuss progress in the application of peptide display, library selection, and directed evolution technologies toward engineering vectors based on retrovirus, adeno-associated virus, and adenovirus that are capable of targeted delivery to specific cell types. In addition to creating biomedically useful products, these approaches have future potential to yield novel insights into viral structure-function relationships.

The Tasmanian devil (Sarcophilus harrisii) is currently threatened by an emerging wildlife disease, devil facial tumour disease. The disease is decreasing devil numbers dramatically and may lead to the extinction of the species. At present, nothing is known about the immune genes or basic immunology of the devil. In this study, we report the construction of the first genetic library for the Tasmanian devil, a spleen cDNA library, and the isolation of full-length MHC Class I and Class II genes. We describe six unique Class II beta chain sequences from at least three loci, which belong to the marsupial Class II DA gene family. We have isolated 13 unique devil Class I sequences, representing at least seven Class I loci, two of which are most likely non-classical genes. The MHC Class I sequences from the devil have little heterogeneity, indicating recent divergence. The MHC genes described here are most likely involved in antigen presentation and are an important first step for studying MHC diversity and immune response in the devil.

Cellular differentiation is an essential characteristic of cells required for the formation of multicellular organisms. However, many unicellular organisms, such as yeast, also differentiate in order to survive in hostile environments. Recent studies suggest that the mechanisms and factors involved in differentiation are not very cell type specific, let alone organism specific (Yamamoto et al. 1999; Hiroi et al. 2002). Schizosaccharomyces pombe, a fission yeast, resembles higher eukaryotes in several ways including genetic structure, cell cycle control, and various signaling pathways. It also undergoes cellular differentiation during sexual development involving conjugation, meiosis, and sporulation. Sexual differentiation is controlled primarily through two signals: nutrient starvation and the presence of mating pheromone. Transcriptional factor Ste11 is required and activates several genes involved in conjugation and meiosis (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Willer et al. 1995). Included are the mei2, rep1, and ste genes (including ste11), and other mating-type genes (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Miyamoto et al. 1994; Sugiyama et al. 1994; Willer et al. 1995). Nitrogen starvation as well as carbon starvation are two major nutrient-depletion signals triggering sexual development in budding yeast. Pat1/Ran1 kinase plays a unique role in the regulation of sexual development, primarily blocking the onset of meiosis until conjugation occurs by inactivating a key triggering factor, Mei2 (Beach et al. 1985; Iino and Yamamoto 1985a,b; Watanabe et al. 1997). Inactivation of Pat1 in haploid cells induces lethal meiosis that can be suppressed through inactivation of the mei2 gene. As Ste11 is required for the expression of mei2 (Sugimoto et al. 1991), factors inhibiting ste11 expression or function would rescue the pat1 inactivated cells. Rcd1 (required for cell differentiation) was identified as a differentiation-controlling factor crucial for nitrogen starvation-induced sexual development in S. pombe by screening a genetic library for suppressors of pat1 lethality (Okazaki et al. 1998). Surprisingly, cells with nonfunctional rcd1 were also shown to be sterile, despite initial assumptions that these strains would have been highly proficient for sexual development. Rcd-1 is essential for nitrogen starvation-invoked induction of ste11 (Okazaki et al. 1998). Paradoxically, overexpression of Rcd-1 rescued pat1 inactivated cells, supposedly through the inhibiting activity of a dominant active mutant of Mei2 (Y. Watanabe and M. Yamamoto, unpubl.). Homologs of the rcd1 gene from S. pombe are quite conserved throughout eukaryotic genomes (Fig. 1A; Okazaki et al. 1998). One homolog, CAF40, was identified as a component of the CCR4–NOT transcription complex in Saccharomyces cerevisiae (Chen et al. 2001). This complex is evolutionarily conserved in eukaryotes and is involved in initiation (Denis and Malvar 1990; Sakai et al. 1992; Collart and Struhl 1993), elongation (Denis et al. 2001), degradation (Tucker et al. 2001), and deadenylation (Tucker et al. 2001) of mRNA, as well as regulation of TFIID activity (Chen et al. 2002). In the CCR4–NOT complex, only NOT1 is essential (Collart and Struhl 1993; Maillet et al. 2000), although it has no identifiable functional domains, apparently acting as a scaffold. CAF40 associates with NOT1 through the latter's N-terminal 1100 residues, although its presence was not required for CCR4–NOT complex formation (Chen et al. 2001). As CAF40 has also been shown to interact with human NOT1, this suggests that hRcd-1 would be a part of the human CCR4–NOT complex (Chen et al. 2001). Figure 1. Rcd-1 sequences. (A) Alignment of the sequences of Rcd-1 from various organisms as indicated (GenBank accession nos.: Homo sapiens, {"type":"entrez-protein","attrs":{"text":"NP_005435","term_id":"4885579"}}NP_005435; S. pombe, {"type":"entrez-protein","attrs":{"text":"NP_594984","term_id":"19115896"}} ... Mammalian Rcd-1 was investigated for its functional similarity to the yeast homologs using F9 mouse teratocarcinoma cell differentiation, which is regulated by retinoic acid (RA) (Hiroi et al. 2002). F9 cells differentiate into primitive endoderm upon treatment with RA (Hogan et al. 1981; Edwards and McBurney 1983). Inhibition of mouse Rcd-1 (mRcd-1) production through the use of antisense oligonucleotides effectively blocked RA-induced differentiation (Hiroi et al. 2002). As well, overexpression of mRcd-1-induced spontaneous differentiation, apparently sensitizing the F9 cells to rather low levels of RA (Hiroi et al. 2002). Rcd-1 is highly expressed in various mammalian tissues. Antibodies against Rcd-1 were used to probe various tissues from adult and embryonic rat, showing the highest levels of expression in the lung, spleen, testes, and thymus of adult rats, while expressed in virtually all tissues of the early rat embryo (Hiroi et al. 2002). Northern blots using human Rcd-1 as a probe showed significant expression in human testes, ovaries, and thymus (Okazaki et al. 1998). mrcd1 was also isolated as an erythropoietin-responsive gene potentially involved in hematopoetic cell development (Gregory et al. 2000). A recent finding reports that mRcd-1 interacts with the c-Myb protein (Haas et al. 2004), which, in turn, is required for the generation of monocytes from a myeloid progenitor (for review, see Friedman 2002). These results are consistent with Rcd-1’s proposed involvement in differentiation control. Despite the increasing knowledge about the role Rcd-1 plays in cellular differentiation, little is known about the mechanisms by which Rcd-1 may exert its influence. Structural information could provide more insight regarding its actual functioning within cells. We now report the crystal structure of the highly conserved region of human Rcd-1 (a region spanning 90% of the full-length protein). The armadillo-repeat-like structure in conjunction with interesting surface features and charge clustering suggest possible means by which Rcd-1 can mediate its effects on cellular differentiation-related gene control.

With the advent of the so-called genomic revolution and improved techniques of DNA analysis, combined with a rapidly vanishing biodiversity, the systematic community has been facing a remarkable—and often neglected—challenge for the past 50 years: to preserve genetic resources issued from research. The preservation and long-term storage of biological specimens’ derived materials (e.g., DNA extracts) and associated data are essential to ensure comparability and reproducibility in all areas of biological research. Natural history museums and herbaria around the world are now in a position to face the exciting and challenging task of preserving the genetic library of life for generations to come. However, the lack and/or poor condition of preservation of molecular vouchers generated from often fragile and rare specimens have been problems too often underestimated or unable to be addressed due to lack of funding or, more pointedly, lack of interest in preservation of these important research materials. The present article does not seek to reiterate the plea for genetic resource collections introduce by Dessauer et al. in 1984 (and more recently by Sheldon, 2001, and Savolainen and Reeves, 2004). It seeks to bring these collections, and the issues of preservation of genetic resources, to the awareness of the systematic biology community through the case study of the American Museum of Natural History new cryogenic repository, the Ambrose Monell Collection for Molecular and Microbial Research (AMCC; website: http://research.amnh.org/ amcc/).

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.

Mark Pagel relishes an analysis of how natural selection riffles through life's immense genetic library.

Surgical research encompasses any research performed in a surgical department, conducted by surgeons, conceived by surgeons, or financed by surgeons. Thus, surgical research is simply medical research that benefits from the views and experience of surgeons as well as from the financial resources of surgical departments. Consequently, surgical research is now in direct competition with all facets of medical research conducted by medical and basic science departments. Given that medical research is increasingly focusing on molecular analysis, the surgical researcher must contend with the rapidly developing field of molecular biology. Embracing molecular biological techniques and embarking on a research plan that involves cloning of a novel gene or cDNA are ambitious steps for any investigator who is not a trained molecular biologist. On the other hand, when successful, the rewards of such a plan are great. Cloning and studying a novel gene, mRNA, and protein will be viewed as novel and unique research, two key ingredients for obtaining research funding. Fortunately, the advent of molecular biology kits and service companies has made this process easier and more expedient. However, it is crucial to understand the principles and methods used to complete the many tasks that must be accomplished before bringing such a research plan to fruition.

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

In this paper we introduce a novel approach to the use of automatically defined functions (ADFs) with the help of a genetic library builder (GLiB). The new technique uses the two mutation operators of GLiB to automatically create subpopulations of ADFs during evolution, where these are termed evolution-defined functions (EDFs). Our approach consists of dynamically specifying separate subpopulations for each identified ADF, where a further population of programs uses individuals from these subpopulations during evaluations. Using a multiplexer problem and two classification tasks we compare a number of existing methods with this co-evolutionary approach. It is shown that dynamically creating ADF subpopulations (according to worth) proves more beneficial than specifying them a priori. It is also shown that the approach performs better than existing approaches — GP with ADFs and GP with GLiB — at all three tasks. Further, we extend the approach to allow the number of EDFs to emerge during the course of evolution, removing the need to specify how many functions are available a priori.

A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the antiPC on the fiber, it can be reacted with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.

Three cloned Klebsiella pneumoniae nitrogenase gene probes were used to perform genomic Southern hybridization analysis and to screen an Anabaena variabilis-Charon 30 genetic library for Anabaena nitrogenase genes. One of ten phage clones that were isolated using a Klebsiella nifHD gene probe was studied in detail. This clone (ABN 10) contains a 9 kb Eco R1 fragment which was observed in genomic Southerns. ABN 10 does not hybridize with a Klebsiella nifK gene probe. A 2.8 kb Hind III fragment that lies within the 9 kb Eco R1 fragment contains the linked Anabaena nifH and nifD genes; the nifK gene lies at least 4 kb away. Partial sequencing of the nifH and nifD genes confirmed their identity and showed strong conservation of amino acid sequence in comparison to Anabaena 7120, Klebsiella pneumoniae, Azotobacter vinlandii, and Clostridium pasteurianum. When the cloned Anabaena nif genes were put into Klebsiella cells on plasmids, they were not expressed, suggesting that the Anabaena promoters are not recognized by Klebsiella transcription factors.

The genetic library of Tribolium castaneum contains some 125 mutations described so far. Close to 50 of these are sex-linked (recessive or dominant) or autosomal (recessives, semi-dominants or dominants). Fourteen of the 50 mutations affecting a wide variety of body parts were examined in the hybrids resulting from crosses between T. castaneum (Herbst) and T. freemani Hinton, a recently rediscovered species of the castaneum species group. The behavior of the mutations in the hybrids was identical in phenotypic expression and action as those previously observed and reported in T. castaneum , attesting to the similar genetics of the two species. Where hybridization of beetles is possible, this technique provides a means of identifying wild types alleles in common, i.e. homologous, in the two species.

The gtfA gene of Streptococcus mutons GS-5, which encodes a 55-kDa glucosyltransferase has been isolated from a genetic library in an Escherichia coli-Bacillus subtilis shuttle vector, pMK3. The construction containing the gene enables E. coli JM83 and a sucrase-deficient mutant of B. subtilis to grow on sucrose as the sole carbohydrate source. The gene is expressed under its own control in both organisms. The level of biochemical activity detectable in B. subtilis carrying the clone is approx. 50% ofthat found in E. coli harboring the same construction. In Bacillus, the gene is expressed through exponential and stationary phases of growth with a decrease in activity as the culture enters stationary phase, corresponding to increases in intracellular protease levels. The enzyme produced in E. coli or B. subtilis harboring the cloned gene is identical to the enzyme produced by S. mutans GS-5 as determined by migration in native polyacrylamide gels.

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.

A genetic library of Streptococcus mutans GS-5, constructed in an Escherichia coli plasmid vector, was screened for cells which could utilize sucrose as the sole carbon and energy source. The recombinant plasmid pFRU1, containing a 4.2-kilobase pair insert of S. mutans DNA, was shown to confer this phenotype. Further characterization of the gene product encoded by pFRU1 revealed that the enzyme was a beta-D-fructosidase with the highest specificity for the beta (2----6)-linked fructan polymer levan. The enzyme could also hydrolyze inulin [beta (2----1)-linked fructan], sucrose, and raffinose with 34, 21, and 12%, respectively, of the activity observed for levan. The gene (designated fruA) appeared to be expressed under its own control in E. coli, as judged by the lack of influence on gene product activity of induction or repression of the beta-galactosidase promoter adjacent to the insertion site on the cloning vector. The protein was purified to homogeneity, as judged by silver staining of purified protein in denaturing and reducing conditions in polyacrylamide gels, from sonic lysate of E. coli, as well as from culture supernatants of S. mutans GS-5 grown in a chemostat at low dilution rate with fructose as the sole carbohydrate source. Both purified proteins had an apparent molecular mass of 140,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were immunologically related and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as determined by Western blotting with antisera raised against the cloned gene product, and were identical in all physical and biochemical properties tested. The pH optimum of the enzyme acting on fructan polymers was 5.5, with a significant amount of activity remaining at pH 4.0. The optimum pH for sucrose degradation was broader and lower, with a peak at approximately 4.5. Enzyme activity was inhibited almost completely by Hg2+ and Ag2+, inhibited partially by Cu2+, not inhibited by fluoride ion or Tris, and slightly stimulated by Mn2+ and Co2+. Fructan polymers were attacked exohydrolytically by the enzyme, fructose being the only product released. With sufficient time, both levan and inulin were degraded to completion, with no evidence of product inhibition.

In the past decade, the fundamental importance and therapeutic potential of costimulatory signals for lymphocyte activation have spurred a large amount of work in immunology, infection, cancer, autoimmune diseases, etc. However, the mechanisms behind T cell costimulation remain unclear, partly due to the lack of suitable techniques. There is an urgent need for functional genomic research to develop comprehensive approaches to direct identification of protein–protein interactions that are dependent on the posttranslational modification of one component of the complex, particularly in the field of T cell immunology. Using inducible costimulator (ICOS) as a model, we failed to find any proteins that associated with the cytoplasmic tail of ICOS by the yeast two-hybrid approach. Therefore, we have developed a new yeast three-hybrid system that facilitates the rapid screening of cDNA libraries to find signaling molecules that interact with phosphorylated T cell costimulatory receptors. We demonstrate the utility of this technique to detect the interaction between ICOS and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The p85 unit of PI3K is the only signaling molecule identified so far that interacts with ICOS. This system may be of great help in dissecting the mechanisms of T cell costimulation and could be applied to other receptors.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for α(1→6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

The idea that there is an identifiable set of boundaries, beyond which anthropogenic change will put the Earth system outside a safe operating space for humanity, is attracting interest in the scientific community and gaining support in the environmental policy world. Rockstrom et al. (2009) identify nine such boundaries and highlight biodiversity loss as being the single boundary where current rates of extinction put the Earth system furthest outside the safe operating space. Here we review the evidence to support a boundary based on extinction rates and identify weaknesses with this metric and its bearing on humanity's needs. While changes to biodiversity are of undisputed importance, we show that both extinction rate and species richness are weak metrics for this purpose, and they do not scale well from local to regional or global levels. We develop alternative approaches to determine biodiversity loss boundaries and extend our analysis to consider large-scale responses in the Earth system that could affect its suitability for complex human societies which in turn are mediated by the biosphere. We suggest three facets of biodiversity on which a boundary could be based: the genetic library of life; functional type diversity; and biome condition and extent. For each of these we explore the science needed to indicate how it might be measured and how changes would affect human societies. In addition to these three facets, we show how biodiversity's role in supporting a safe operating space for humanity may lie primarily in its interactions with other boundaries, suggesting an immediate area of focus for scientists and policymakers.