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Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systemsin vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery ofin vitroassays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, andin vivoinCaenorhabditischemosensory neurons,Drosophilalarval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combiningin vivoimaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activityin vivoand may find widespread applications for cellular imaging in general.

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p -acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR™ (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5–4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known.

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.

The small soil nematode Caenorhabditis elegans has only 302 neurons in its entire nervous system, so it is possible to analyse the functions of individual neurons in the animal's behaviour. We are using behavioural, cellular and genetic analyses of chemotactic responses to find out how olfactory behaviour patterns are generated and regulated. Single chemosensory neurons in C. elegans can recognize several different attractive odorants that are distinguished by the animal. Distinct sets of chemosensory neurons detect high and low concentrations of a single odorant. Odorant responses adapt after prolonged exposure to an odorant; this adaptation is odorant specific and reversible. Mutants with defects in odorant responses have been identified. Some genes appear to be necessary for the development or function of particular kinds of sensory neurons. Other genes have effects that suggest that they participate in odorant reception or signal transduction.

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.

Clostridium difficile ( C. difficile ) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca 2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca 2+ -dependent fashion. Millimolar Ca 2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca 2+ in the sensitization of toxin A/cell-surface interactions.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the ∼120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes ∼13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

rough (ro) encodes a homeobox transcription factor required for proper specification of photoreceptor cells R2 and R5 in Drosophila eye development. To identify the transcriptional targets through which ro acts to specify the R2/R5 neuronal sub-type, we screened enhancer trap lines expressed in developing photoreceptors for those whose expression patterns were altered when ro function was inactivated. In this way we identified two potential ro targets, which are also targets of the zinc finger transcription factor glass (gl) . We also identified an enhancer trap line that exhibits altered morphogenetic furrow expression in a ro mutant background. Finally, we have molecularly characterized an enhancer trap line, AE33, that was identified in earlier screens as a target of both ro and gl (Freeman et al., 1992; Treisman and Rubin, 1996). The transcript interrupted by AE33 shares similarity with the mammalian vasodilator-stimulated phosphoprotein ( VASP ), a substrate for cAMP- and cGMP-dependent protein kinases that is associated with actin filaments, focal adhesions, and dynamic membrane regions (Haffner et al., 1995) with enabled (ena), a substrate of the Drosophila Abl tyrosine kinase (Gertler et al., 1995) and with two human Expressed Sequence Tags (ESTs).

The Drosophila homeo box gene rough is required in photoreceptor cells R2 and R5 for normal eye development. We show here that rough protein expression is limited to a subset of cells in the developing retina where it is transiently expressed for 30-60 hr. The rough protein is first expressed broadly in the morphogenetic furrow but is rapidly restricted to the R2, R3, R4, and R5 precursor cells. Ubiquitous expression of rough under the control of the hsp70 promoter in third-instar larvae suppresses the initial steps of ommatidial assembly. Structures derived from other imaginal discs are not affected. Ectopic expression of rough in the R7 precursor, through the use of the sevenless promoter, causes this cell to develop into an R1-6 photoreceptor subtype; however, this cell still requires sevenless function for its neural differentiation. Taken together with previous analyses of the rough mutant phenotype, these results suggest that the normal role of rough is to establish the unique cell identity of photoreceptors R2 and R5.

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.

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 rough mutation in Drosophila disrupts an early stage of ommatidial assembly in the developing eye imaginal disc. Analysis of somatic mosaics suggests that rough gene function is required only in photoreceptors R2 and R5. However, these cells apparently differentiate normally in mutant eye discs and it is the cells that are subsequently added to the ommatidium that behave aberrantly. The rough gene was isolated by P-element transposon tagging, and the mutant can be rescued by transformation with an 8.6 kb genomic fragment. The rough transcription unit is 4.3 kb and consists of three exons that are joined in a 1.3 kb mRNA. The predicted rough protein is 350 amino acids long and contains a homeobox. Taken together, our results suggest that the role of the rough gene product may be to regulate the sending of signals by photoreceptors R2 and R5 to their neighbors in the developing ommatidia.

Two-dimensional polyacrylamide gel electrophoresis has been used to analyze changes in protein content and protein synthesis in three stages of the life cycle of the protozoan parasite Trypanosoma brucei. The stages examined were slender and stumpy mammalian bloodstream forms and procyclic forms, which are analogous to the tsetse fly midgut stage. Two-dimensional gels of 35S-methionine-labeled proteins were examined by autoradiography to analyze newly synthesized protein, and gels were stained with ammoniacal silver to analyze proteins present. Several stage-specific molecules were noted. The most obvious was the variant surface glycoprotein, which was only present in bloodstream forms. Some other proteins were also bloodstream form specific; they had molecular weights of 120,000 and 38,000. Proteins of 52,000, 46,000, 25-30,000, and 16,000 daltons were present both in stumpy forms and procyclics but not in slender-form trypanosomes. Several proteins (molecular weights of 50-70,000, 43,000, 40,000, 26-24,000, 20-25,000, and 15,000) were present only in one of the three stages. One protein, a molecule of about 18,000 daltons present in both slender and stumpy parasites, did not appear to be synthesized in the stumpy stage. In vitro translation products of mRNA purified from the three stages were also examined. The abundance of mRNA encoding a protein of about 40,000 daltons appeared to be greater in slender than in stumpy parasites although the stumpy forms contained more of the protein and synthesized it at a higher rate.

A simple technique for raising specific antisera to protein encoded by cloned genes is described. The procedure involves preparation of an antiserum to Escherichia coli beta-galactosidase and the use of that serum to immunoprecipitate a fusion protein in a crossed immunoelectrophoresis gel followed by immunization with fusion protein precipitin arcs. An antiserum was prepared against protein encoded by an open reading frame in a dispersed repeated DNA sequence found in the protozoan Trypanosoma brucei. This serum recognized a polypeptide doublet of 33.5 and 32.5 kDa on immunoblots prepared from extracts of T. brucei. The method described should be applicable to other investigations where an immunochemical reagent against protein encoded by a cloned gene is desired.

The sevenless gene encodes a cell surface receptor that has protein-tyrosine kinase activity and is expressed in a highly specific and complex pattern in the developing Drosophila eye. We have coupled the sevenless promoter to the reporter gene lacZ and have examined the pattern of beta-galactosidase expression in the developing eyes of transgenic larvae. Our results indicate that the dynamic pattern of sevenless protein expression is regulated transcriptionally. Promoter sequences located 5' of the coding region are insufficient for the wild-type level of gene expression but appear to be able to confer the correct pattern of expression. In contrast, enhancer sequences within the body of the gene can confer both the correct pattern and a normal level of expression on either the sevenless promoter or heterologous promoters. Thus the complex pattern of sevenless expression is redundantly encoded within proximal promoter sequences and enhancer elements internal to the gene but relies on these enhancer sequences for correct quantitative expression.

Different olfactory cues elicit distinct behaviors such as attraction, avoidance, feeding, or mating. In the nematode C. elegans, these cues are sensed by a small number of olfactory neurons, each of which expresses several different odorant receptors. The type of behavioral response elicited by an odorant could be specified by the olfactory receptor or by the olfactory neuron in which the receptor is activated. The attractive odorant diacetyl is detected by the receptor protein ODR-10, which is normally expressed in the AWA olfactory neurons. The repulsive odorant 2-nonanone is detected by the AWB olfactory neurons. Transgenic animals that express ODR-10 in AWB rather than AWA avoid diacetyl, while maintaining qualitatively normal responses to other attractive and repulsive odorants. Animals that express ODR-10 simultaneously in AWA and AWB have a defective response to diacetyl, possibly because of conflicting olfactory inputs. Thus, an animal's preference for an odor is defined by the sensory neurons that express a given odorant receptor molecule.

The mechanism controlling transcription at several telomeric expression sites for variable surface glycoprotein (VSG) genes in Trypanosoma brucei is unknown. Most VSG genes in expression sites have a region 5′ of the gene lacking restriction enzyme sites. This ‘barren region’ is involved in recombination events which replace the VSG gene with a copy of a different, non-telomeric, VSG gene leading to a switch in VSG expression. Alterations in the barren region have been considered as possible modulators of expression of the adjacent VSG gene in other switching events where no gene replacement occurs. The expressed copy of the ILTat 1.3 VSG gene remains in its expression site, on a 160 kilobase (kb) chromosome, in trypanosomes not expressing the ILTat 1.3 VSG. Here we report the complete sequence of the barren region adjacent to this gene, determined both from trypanosomes expressing the gene and from those that are not. The sequence is identical whether or not the ILTat 1.3 VSG gene is expressed. This confirms that alterations in the barren region are not involved in modulation of expression of the gene, as suggested by restriction enzyme mapping. Sequence data from the 5′ flanking region of a second telomeric gene copy on an 80 kb minichromosome, and from the ILTat 1.3 expression site after replacement of the ILTat 1.3 gene by another gene from a minichromosome, provide evidence that telomeric VSG genes on minichromosomes are also flanked by long repeat arrays, and that these arrays are involved in inter-telomeric gene replacements as well as replacements by non-telomeric genes.