Algorithms for sequence analysis via mutagenesis

MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Pathological glial tau accumulations in neurodegenerative disease: review and case report

Abnormal deposits of tau protein accumulate in glia in many neurodegenerative diseases. This suggests that in some instances the disease process may target glial tau, with neuronal degeneration a secondary consequence of this process. In this report, we summarize the pattern of glial tau pathology in various neurodegenerative disorders and add original findings from a case of sporadic frontotemporal dementia that exhibits astrocytic tau pathology. The neurodegenerative diseases span the spectrum of relative neuronal and glial tau involvement, from disorders affecting only neuronal tau to those in which abnormal tau deposits are found only in glia. From this, we conclude that glial tau can be a primary target of the disease process, and that this can lead to neuronal degeneration.     

HABITAT ACOUSTICS OF A NEOTROPICAL LOWLAND RAINFOREST

ABSTRACT

The acoustic characteristics of an Amazonian lowland rain forest study site in southern Venezuela was analysed to determine environmental constraints upon acoustic communication. Signal degradation was measured by conducting transmission experiments at different heights above ground level. Measurements of ambient noise served to determine possible communication distances for various times of day, heights above ground level and frequencies. “Sound windows” for acoustic long-range communication were found for low frequencies, calling heights in the midstorey and calling in the morning or during the night. Sound attenuation was affected by height and frequency but not by time of day. Background noise varied remarkably with time of day and frequency and had a greater impact on communication distance than signal attenuation.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.     

Phylogeny and evolution of corambid nudibranchs (Mollusca: Gastropoda)

Organismic diversity, as well as distributional and ecological patterns, can be fully understood in an evolutionary framework only. Reliable phylogenetic trees are required to ‘read history’, but are not yet available for most marine invertebrate groups. Molecular systematics offers an enormous potential, but still fails for ‘all‐species approaches’ on groups with species that are rare or occur in remote areas only, simply because there is no easily collectable material available for sequence analyses. Exploring morphologically aberrant corambid nudibranch gastropods as a case study, we assess whether or not morphology‐based phylogenetic analyses can fill this gap and produce a tree that allows a detailed view on evolutionary history. Morphology‐based parsimony analysis of corambids and potential relatives resulted in a well‐resolved and remarkably robust topology. As an offshoot of kelp‐associated onchidoridid ancestors, and obviously driven by the heterochronic shortening of life cycles and morphological juvenilization in an ephemeral habitat, the ancestor of corambids originated in cool northern Pacific coastal waters. A basal clade (the genus Loy) diverged there, adapting to live on soft bottoms under successive reversals of paedomorphic traits. The more speciose Corambe lineage radiated preying upon short‐lived encrusting bryozoa in a high‐energy kelp environment. Selection favoured transformation of the mantle into a cuticle‐covered shield, and successive paedomorphic translocations of dorid anal gills to the protected ventral side of the body, where compensatory, multiple gills evolved. Corambe species probably first colonized tropical American seas, and then radiated in worldwide temperate waters: this is explained by the excellent long‐distance dispersal abilities afforded by rafting on kelp, with the subsequent divergence of colonizers in allopatry. The competitive coexistence of Corambe pacifica MacFarland & O'Donoghue, 1929 and Corambe steinbergae (Lance, 1962) off California is the result of independent colonization events. The closing of the Isthmus of Panama separated the latter species from a flock that have radiated within warm Atlantic waters since then. Our case study shows that morphological structures, if investigated in depth, bear the potential for an efficient phylogenetic analysis of groups that are still elusive to molecular analyses. Tracing character evolution and integrating a wide range of geographic, biological, and ecological background information allowed us to reconstruct an evolutionary scenario for corambids that is detailed and plausible, and can be tested by future molecular approaches. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 585–604.