Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.     

An efficient model development strategy for bioprocesses based on neural networks in macroscopic balances: Part II

There is a need for efficient modeling strategies which quickly lead to reliable mathematical models that can be applied for design and optimization of (bio)-chemical processes. The serial gray box modeling strategy is potentially very efficient because no detailed knowledge is needed to construct the white box part of the model and because covenient black box modeling techniques like neural networks can be used for the black box part of the model. This paper shows for a typical biochemical conversion how the serial gray box modeling strategy can be applied efficiently to obtain a model with good frequency extrapolation properties. Models with good frequency extrapolation properties can be applied under dynamic conditions that were not present during the identification experiments. For a given application domain of a model, this property can be used to considerably reduce the number of identification experiments. The serial gray box modeling strategy is demonstrated to be successful for the modeling of the enzymatic conversion of penicillin G In the concentration range of 10-100 mM and temperature range of 298-335 K. Frequency extrapolation is shown by using only constant temperatures in the (batch) identification experiments, while the model can be used reliable with varying temperatures during the (batch) validation experiments. No reliable frequency extrapolation properties could be obtained for a black box model, and for a more knowledge-driven white box model reliable frequency extrapolation properties could only be obtained by incorporating more knowledge in the model. Copyright 1999 John Wiley & Sons, Inc.     

Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.      

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

LRV1 viral particles in Leishmania guyanensis contain double-stranded or single-stranded RNA.

The 32-nm-diameter spherical viral particles found in the cytoplasm of Leishmania guyanensis CUMC1-1A sediment at 130S and have a buoyant density of approximately 1.4 g/ml in cesium chloride gradients. These particles contain a 5.3-kb double-stranded RNA, while single-stranded RNA that corresponds to the viral positive strand is associated with less-dense particles. These results suggest a conservative and sequential mode of LRV1 viral RNA replication that is exemplified by the ScV L-A virus of yeast.     

Ploidy changes associated with disruption of two adjacent genes on Leishmania major chromosome 1

Leishmania major Friedlin (LmjF) is a kinetoplastid protozoan whose genomic sequence has been recently elucidated. About 60% of the identified genes do not have a known function, and many are trypanosomatid-specific. Here we characterise two adjacent genes from LmjF chromosome 1 (chr1): LmjF01.0750, which encodes a predicted protein with a serine/threonine protein kinase motif and LmjF01.0760, which encodes a product with no similarity to other known proteins. Orthologues of both genes are present in Trypanosoma cruzi, but neither occur in Trypanosoma brucei. We have mapped polyadenylation and spliced-leader acceptor sites for both genes, and show that they differ between Leishmania species. Attempts to generate null mutants of LmjF01.0750 by homologous recombination were unsuccessful and led to the apparent triploidy of the entire genome, suggesting that it is an essential gene. Interestingly, at least two copies of LmjF01.0750 are required for cell survival. Further evidence of genome plasticity in Leishmania was provided by changes in chr1 copy number that occurred during in vitro growth of wild-type LmjF promastigotes and following replacement of a single copy of LmjF01.0760.