Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS

Separation of synthetic deoxyribo-ligonucleotides by thin layer chromatography

NRC publication: Ye

Photomorphogenesis

As photoautotrophs, plants are exquisitely sensitive to their light environment. Light affects many developmental and physiological responses throughout plants' life histories. The focus of this chapter is on light effects during the crucial period of time between seed germination and the development of the first true leaves. During this time, the seedling must determine the appropriate mode of action to best achieve photosynthetic and eventual reproductive success. Light exposure triggers several major developmental and physiological events. These include: growth inhibition and differentiation of the embryonic stem (hypocotyl); maturation of the embryonic leaves (cotyledons); and establishment and activation of the stem cell population in the shoot and root apical meristems. Recent studies have linked a number of photoreceptors, transcription factors, and phytohormones to each of these events

Phototropism: Mechanism and Outcomes

Plants have evolved a wide variety of responses that allow them to adapt to the variable environmental conditions in which they find themselves growing. One such response is the phototropic response - the bending of a plant organ toward (stems and leaves) or away from (roots) a directional blue light source. Phototropism is one of several photoresponses of plants that afford mechanisms to alter their growth and development to changes in light intensity, quality and direction. Over recent decades much has been learned about the genetic, molecular and cell biological components involved in sensing and responding to phototropic stimuli. Many of these advances have been made through the utilization of Arabidopsis as a model for phototropic studies. Here we discuss such advances, as well as studies in other plant species where appropriate to the discussion of work in Arabidopsis

Flower Development

Flowers are the most complex structures of plants. Studies of Arabidopsis thaliana, which has typical eudicot flowers, have been fundamental in advancing the structural and molecular understanding of flower development. The main processes and stages of Arabidopsis flower development are summarized to provide a framework in which to interpret the detailed molecular genetic studies of genes assigned functions during flower development and is extended to recent genomics studies uncovering the key regulatory modules involved. Computational models have been used to study the concerted action and dynamics of the gene regulatory module that underlies patterning of the Arabidopsis inflorescence meristem and specification of the primordial cell types during early stages of flower development. This includes the gene combinations that specify sepal, petal, stamen and carpel identity, and genes that interact with them. As a dynamic gene regulatory network this module has been shown to converge to stable multigenic profiles that depend upon the overall network topology and are thus robust, which can explain the canalization of flower organ determination and the overall conservation of the basic flower plan among eudicots. Comparative and evolutionary approaches derived from Arabidopsis studies pave the way to studying the molecular basis of diverse floral morphologies