Improving catalytic function by ProSAR-driven enzyme evolution

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.     

Expression and purification of soluble monomeric streptavidin in Escherichia coli

We recently reported the engineering of monomeric streptavidin (mSA) for use in monomeric detection of biotinylated ligands. Although mSA can be expressed functionally on the surface of mammalian cells and yeast, the molecule does not fold correctly when expressed in Escherichia coli. Refolding from inclusion bodies is cumbersome and yields a limited amount of purified protein. Improving the final yield should facilitate its use in biotechnology. We tested the expression and purification of mSA fused to GST, MBP, thioredoxin, and sumo tags to simplify its purification and improve the yield. The fusion proteins can be expressed solubly in E. coli and increase the yield by more than 20-fold. Unmodified mSA can be obtained by proteolytically removing the fusion tag. Purified mSA can be immobilized on a solid matrix to purify biotinylated ligands. Together, expressing mSA as a fusion with a solubilization tag vastly simplifies its preparation and increases its usability in biotechnology.     

Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance

Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a suitable microarray-based protocol for the detection of point mutations in M. tuberculosis genes associated with drug resistance. We initially developed a conventional, oligonucleotide microarray protocol and used it to detect and identify on a single microarray slide a number of point mutation-containing rpoB and katG gene target sequences. However, the occurrence of some non-specific hybridization led us to the development of an improved protocol based on allele specific PCR combined with tags/anti-tags and microarrays. This protocol was evaluated by detecting point mutations in M. tuberculosis katG and rpoB gene templates produced by recombinant PCR. The methodology allowed sequences containing single point mutations to be readily distinguished from wild type sequences. The data obtained with the improved protocol had strong and specific signals and relatively low amounts of non-specific hybridization. We successfully used this protocol to detect and identify (<8 h) a number of clinically relevant point mutations in the rpoB, katG and rpsL genes of M. tuberculosis clinical isolates. Our allele specific PCR/tags and anti-tags/microarray protocol has several advantages over our conventional oligonucleotide microarray protocol, and it may have broad applications for point mutation detection.     

Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production.

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.     

The relationships of the Bornean Bristlehead(Pityriasis gymnocephala) and the Black-collared Thrush(Chlamydochaera jefferyi)

Summary The taxonomic relationships of two Bornean endemics,Pityriasis gymnocephala andChlamydochaera jefferyi, have been considered to be uncertain. DNA-DNA hybridization was used to compare the single-copy sequences of each of these two species with other passerine taxa. The DNA data support other evidence indicating thatPityriasis is a cracticine corvoid, most closely related to the Australo-Papuan generaCracticus, Gymnorhina, Strepera, andPeltops. Chlamydochaera is a typical turdine thrush, closely related toTurdus. This supports and confirms the morphological evidence presented byAmes (1975).Chlamydochaera is not related to the cuckoo-shrikes (Campephaga, etc.) of the Tribe Oriolini.

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Die systematische Stellung des Warzenkopfes (Pityriasis gymnocephala) und des Fruchtpickers(Chlamydochaera jefferyi)

Zusammenfassung Die systematische Stellung der beiden auf Borneo endemischer Arten galt bisher als unsicher. DNS-Kreuzung wurde durchgeführt, um die Sequenzen eines Einzelstrangs jeder der beiden Arten mit anderen Singvogeltaxa zu vergleichen. Die DNS-Untersuchungen unterstützen andere Hinweise auf nähere Beziehung vonPityriasis zu den Cracticidae des australo-papuanischen Faunenreiches(Cracticus, Gymnorhina, Strepera, Peltops). Chlamydochaera ist eine typische Drossel, nahe verwandt mitTurdus. Dieses Ergebnis bestätigt die vonAmes (1975) erhobenen morphologischen Befunde.Chlamydochaera ist mit den Stachelbürzlern [Campephaga usw.), Tribus Oriolini, also nicht näher verwandt.

     

Spatial and Temporal Variation of Seed Rain in the Canopy and on the Ground of a Tropical Cloud Forest

Spatial and temporal patterns of seed rain impact plant fitness, genetic and demographic structure of plant populations, and species' interactions. Because plants are sessile, they rely on biotic and abiotic dispersal agents to move their seeds. The relative importance of these dispersal agents may shift throughout the year. In tropical forests, seed dispersal of epiphytes constitutes a major but hitherto unknown portion of seed rain ecology. For the first time, we report on patterns of seed rain for both epiphytic and terrestrial plants across an entire year in a Neotropical montane forest. To examine seed rain, we placed traps in the canopy and on the ground. We analyzed seed dispersal syndrome (bird, mammal, wind) and plant habit (epiphyte, liana, shrub, small tree, large tree) across all seasons of the year (dry, misty, wet). We found that the community of species collected in canopy traps was significantly different from the community in ground traps. Epiphytes were the most common plant habit found in canopy traps, while large trees were most common in ground traps. Species with bird‐dispersed seeds dominated all traps. Species richness was significantly higher during the dry season in ground traps, but did not vary across seasons in canopy traps. Our results highlight the distinct seed rain found in the canopy and on the ground and underscore the importance of frugivores for dispersing both arboreal and terrestrial plants in tropical ecosystems.     

Molecular homology and DNA hybridization

Summary We reviewed the concept of homology, which can broadly be defined as a correspondence between characteristics that is caused by continuity of information (Van Valen 1982). The concept applies widely in molecular biology when correspondence is taken to mean a genetic relationship resulting from a unique heritable modification of a feature at some previous point in time. Such correspodence can be established for features within a single organism as well as between organisms, making paralogy a valid form of molecular homology under this definition. Molecular homology can be recognized at a variety of organizational levels, which are intedependent. For example, the recognition of homology at the site level involves a statement of homology at the sequence level, and vice versa. This hierarchy, the potential for nonhomologous identity at the site level, and such processes as sequence transposition combine to yield a molecular equivalent to complex structural homology at the anatomical level. As a result, statements of homology between heritable units can involve a valid sense of percent homology.We analyzed DNA hybridization with respect to the problems of recognizing homology and using it in phylogenetic inference. Under a model requiring continuous divergence among compared sequences, DNA hybridization distances embed evolutionary hierarchy, and groups inferred using pairwise methods of tree reconstruction are based on underlying patterns of apomorphic homology. Thus, symplesiomorphic homology will not confound DNA hybridization phylogenies. However, nonhomologous identities that act like apomorphic homologies can lead to inaccurate reconstructions. The main difference between methods of phylogenetic analysis of DNA sequences is that parsimony methods permit hypotheses of nonhomology, whereas distance methods do not.This article was presented at the C.S.E.O.L. Conference on DNA-DNA Hybridization and Evolution, Lake Arrowhead, California, May 11–14, 1989     

Glial perspectives of metabolic states during cerebral hypoxia--calcium regulation and metabolic energy

Cooperation between astrocytes and neurons is a unique interaction between two highly specialized cell types of the brain. Therefore, lack of nutrient supply during ischemia requires tight coordination of metabolism between astrocytes and neurons to keep the brain functions intact. To understand the impact of energy limitation on astrocytes, the functions of astrocytes have to be considered: (i) supplementation of neuronal cells, (ii) modulation of the extracellular milieu, mainly of the glutamate level, and (iii) elimination of reactive oxygen species (ROS). In cultured astrocytes and neurons inhibition of oxidative phosphorylation, using rotenone, was tested. Interestingly, this had only a negligible effect on Ca2+ homeostasis in astrocytes, even in combination with a severe glutamate stress. In contrast, in neurons glutamate in the presence of rotenone induced Ca2+ deregulation. Ca2+ homeostasis is very critical for cell survival. A massive and prolonged Ca2+ rise will lead to deregulation of many processes in such a way that the cells affected can hardly survive. Ca2+ homeostasis depends on the energy-consuming processes, which maintain the steep gradient between intracellular and extracellular Ca2+ concentration. Deprivation of oxygen and glucose during ischemia leads to a depletion of ATP in the brain, due to inhibited glycolytic and mitochondrial activity, whereas energy-consuming processes like ion pumps drain the ATP pools. On the other hand, specific mechanisms can protect brain structures against the massive insult of ischemia. Glycogen, stored in astrocytes, can maintain both neurons and astrocytes alive during short limitation of oxygen and glucose. Moreover, astrocytes can fuel ATP generation by providing lactate for neurons.     

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.