Improved Coated Vesicle Isolation Allows Better Characterization of Clathrin Polypeptides

Postmicrosomal pellets from plant sources are grossly contaminatedwith nbosomes. Previous purifications of clathrin coated vesicles(CCV) from such subcellular fractions have therefore often involvedan RNase treatment. Performed at 30°C, this step inherentlycarries with it the dangers of proteolysis. We document herea method for CCV isolation which avoids this. Through the inclusionof suitable antiproteases in the homogenizing and subsequentisolation media, we have also been able to improve the qualityof CCV recovered from plant tissues. As a result we have tentativelybeen able to identify clathrin light chains from zucchini hypocotyland pea cotyledon CCV. Similar to light chains from bovine brainthese polypeptides are heat stable, can be solubilized fromneutralized TCA precipitates, bind calcium and clathrin heavychains. However, in contrast to brain CCV the two light chainsof plant CCV are some 10 kDa heavier. Key words: Antiproteases, Ca²⁺-binding, clathrin coated vesicles, clathrin heavy chains, clathrin light chain(s), heat stability, pea cotyledons, RNase, zucchini hypocotyls     

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp.     

Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies

Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, Z_Ca, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the Z_Ca–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.     

小鼠染色体工程的研究进展

以染色体大片段的删除和重排为主要特征的小鼠染色体工程逐渐成为一种大规模研究小鼠基因功能的重要手段。这里重点介绍了小鼠染色体工程的最新研究进展。     

Broad Spectrum of Mimiviridae Virophage Allows Its Isolation Using a Mimivirus Reporter

The giant virus Mimiviridae family includes 3 groups of viruses: group A (includes Acanthamoeba polyphaga Mimivirus), group B (includes Moumouvirus) and group C (includes Megavirus chilensis). Virophages have been isolated with both group A Mimiviridae (the Mamavirus strain) and the related Cafeteria roenbergensis virus, and they have also been described by bioinformatic analysis of the Phycodnavirus. Here, we found that the first two strains of virophages isolated with group A Mimiviridae can multiply easily in groups B and C and play a role in gene transfer among these virus subgroups. To isolate new virophages and their Mimiviridae host in the environment, we used PCR to identify a sample with a virophage and a group C Mimiviridae that failed to grow on amoeba. Moreover, we showed that virophages reduce the pathogenic effect of Mimivirus (plaque formation), establishing its parasitic role on Mimivirus. We therefore developed a co-culture procedure using Acanthamoeba polyphaga and Mimivirus to recover the detected virophage and then sequenced the virophage''s genome. We present this technique as a novel approach to isolating virophages. We demonstrated that the newly identified virophages replicate in the viral factories of all three groups of Mimiviridae, suggesting that the spectrum of virophages is not limited to their initial host.     

Shape-Induced Asymmetric Diffusion in Dendritic Spines Allows Efficient Synaptic AMPA Receptor Trapping

Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.     

Sex Chromosome Translocations in the Evolution of Reproductive Isolation

Haldane's rule states that in organisms with differentiated sex chromosomes, hybrid sterility or inviability is generally expressed more frequently in the heterogametic sex. This observation has been variously explained as due to either genic or chromosomal imbalance. The fixation probabilities and mean times to fixation of sex-chromosome translocations of the type necessary to explain Haldane's rule on the basis of chromosomal imbalance have been estimated in small populations of Drosophila melanogaster. The fixation probability of an X chromosome carrying the long arm of the Y(X·Y^L) is approximately 30% greater than expected under the assumption of no selection. No fitness differences associated with the attached Y^L segment were detected. The fixation probability of a deficient Y chromosome is 300% greater than expected when the X chromosome contains the deleted portion of the Y. It is suggested that sex-chromosome translocations may play a role in the establishment of reproductive isolation.     

Shape-Induced Asymmetric Diffusion in Dendritic Spines Allows Efficient Synaptic AMPA Receptor Trapping

Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.     

Engineering the Drosophila Genome: Chromosome Rearrangements by Design

We show that site-specific recombination can be used to engineer chromosome rearrangements in Drosophila melanogaster. The FLP site-specific recombinase acts on chromosomal target sites located within specially constructed P elements to provide an easy screen for the recovery of rearrangements with breakpoints that can be chosen in advance. Paracentric and pericentric inversions are easily recovered when two elements lie in the same chromosome in opposite orientation. These inversions are readily reversible. Duplications and deficiencies can be recovered by recombination between two elements that lie in the same orientation on the same chromosome or on homologues. We observe that the frequency of recombination between FRTs at ectopic locations decreases as the distance that separates those FRTs increases. We also describe methods to determine the absolute orientation of these P elements within the chromosome. The ability to produce chromosome rearrangements precisely between preselected sites provides a powerful new tool for investigations into the relationships between chromosome arrangement, structure, and function.     

Microchromosomes Exhibit Distinct Features of Vertebrate Chromosome Structure and Function with Underappreciated Ramifications for Genome Evolution

Microchromosomes are common yet poorly understood components of many vertebrate genomes. Recent studies have revealed that microchromosomes contain a high density of genes and possess other distinct characteristics compared with macrochromosomes. Whether distinctive characteristics of microchromosomes extend to features of genome structure and organization, however, remains an open question. Here, we analyze Hi-C sequencing data from multiple vertebrate lineages and show that microchromosomes exhibit consistently high degrees of interchromosomal interaction (particularly with other microchromosomes), appear to be colocalized to a common central nuclear territory, and are comprised of a higher proportion of open chromatin than macrochromosomes. These findings highlight an unappreciated level of diversity in vertebrate genome structure and function, and raise important questions regarding the evolutionary origins and ramifications of microchromosomes and the genes that they house.     

Collaboration between NDH and KEA3 Allows Maximally Efficient Photosynthesis after a Long Dark Adaptation

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K⁺ Efflux Antiporter3 (KEA3) is a putative thylakoid H⁺/K⁺ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m⁻² s⁻¹ after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m⁻² s⁻¹, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO₂ fixation was delayed after overnight dark adaptation.

Photosynthesis consists of two sets of reactions, the light reactions and the Calvin-Benson cycle. It takes place in the chloroplast and fixes CO₂ into organic compounds using solar energy. In the light reactions, the absorption of photons activates electron transport in two photosystems. In linear electron transport (LET), PSII catalyzes the light-dependent oxidation of water, resulting in the release of oxygen and protons (H⁺) in the thylakoid lumen. The water-derived excised electrons are transferred to PSI through the cytochrome (Cyt) b₆f complex and ultimately to NADP⁺, producing NADPH. This electron transport is coupled with the translocation of H⁺ from the stroma to the thylakoid lumen via the quinone cycle at the Cyt b₆f complex, resulting in the formation of a proton concentration gradient across the thylakoid membrane. This ∆pH contributes to the formation of proton motive force (pmf) in addition to the membrane potential formed across the thylakoid membrane (∆ψ) that results from the uneven distribution of ions across the membrane. The pmf energizes ATP synthesis via F_oF₁-ATP synthase in chloroplasts (Kramer et al., 2003; Soga et al., 2017) and thus influences the efficiency of the light reactions.The Calvin-Benson cycle depends on NADPH and ATP produced by the light reactions. To fix a molecule of CO₂ into a carbohydrate, three molecules of ATP and two molecules of NADPH are needed. However, this ratio of ATP to NADPH (1.5) is not satisfied by LET (Shikanai, 2007). Photorespiration, which takes place due to the low specificity of Rubisco, the CO₂-fixing enzyme for CO₂, increases the energetic requirements in terms of ATP, raising the above ratio to 1.67. The additional ATP is thought to be supplied by cyclic electron transport around PSI (CET; Yamori and Shikanai, 2016). In contrast to LET, CET is driven solely by PSI and does not contribute to the net production of reducing power. CET recycles electrons from ferredoxin (Fd) to the plastoquinone (PQ) pool and contributes to the additional generation of ∆pH via the quinone cycle. As a result, CET balances the production ratio of ATP and NADPH. In angiosperms, CET has been proposed to consist of two pathways: the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-like Photosynthetic Phenotype1 (PGRL1) protein-dependent, antimycin A-sensitive pathway and the NADH dehydrogenase-like (NDH) complex-dependent antimycin A-insensitive pathway (Munekage et al., 2004). The NDH complex pumps four protons, coupled with the movement of two electrons, from Fd to PQ, further increasing the efficiency of ∆pH formation (Strand et al., 2017).In addition to ATP synthesis, the ∆pH component of pmf also contributes to the down-regulation of electron transport (Shikanai, 2014). Acidification of the thylakoid lumen triggers the thermal dissipation of excessively absorbed light energy from the PSII antennae, a process that is monitored by nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Müller et al., 2001). Low lumenal pH also down-regulates the activity of the Cyt b₆f complex, slowing down the rate of electron transport toward PSI (Stiehl and Witt, 1969). CET-dependent ∆pH formation is also necessary to induce the down-regulation of electron transport, as indicated by the phenotype of the pgr5 mutant. The Arabidopsis (Arabidopsis thaliana) pgr5 mutant cannot induce thermal dissipation under excessive light conditions (Munekage et al., 2002), suggesting that CET-generated ∆pH plays an important role in providing a sufficiently acidic lumen pH that can trigger NPQ. The pgr5 mutant is also defective in the down-regulation of Cyt b₆f activity, resulting in hypersensitivity of PSI to fluctuating light intensity (Tikkanen et al., 2010). Compared with the physiological function of the PGR5/PGRL1-dependent CET, the contribution of the NDH-dependent CET to photoprotection is somewhat minor, although clear phenotypes have been observed in these mutants at low light intensities and fluctuating light levels (Ueda et al., 2012; Yamori et al., 2015, 2016). Furthermore, the physiological function of the NDH complex has not been fully clarified.Both ∆pH and ∆ψ contribute to pmf, but only ∆pH down-regulates electron transport. To optimize the operation of the accelerator (ATP synthesis) and the brake on electron transport, it is necessary to precisely regulate the ratio of the two pmf components as well as the total size of pmf (Cruz et al., 2001; Kramer et al., 2003). Several channels and antiporters localized to the thylakoid membrane regulate the partitioning of the pmf components (Spetea et al., 2017). K⁺ Efflux Antiporter3 (KEA3) is thought to be an H⁺/K⁺ antiporter localized to the thylakoid membrane (Armbruster et al., 2014; Kunz et al., 2014), although its antiport activity has not been experimentally demonstrated (Tsujii et al., 2019). Based on its structure, topology, and the mutant phenotypes, KEA3 most likely moves H⁺ from the thylakoid lumen while taking up K⁺ as a counter ion. Consequently, KEA3 transforms ∆pH to ∆ψ and is necessary to rapidly relax the down-regulation of electron transport by raising the luminal pH (i.e. by alkalinizing the lumen). The C-terminal domain of KEA3, KTN (K⁺ transport/nucleotide binding), is exposed to the stroma (Wang et al., 2017) and is thought to regulate its activity by monitoring ATP or NADPH levels (Schlosser et al., 1993; Roosild et al., 2002). However, information on the regulation of KEA3 is limited. Armbruster et al. (2014) demonstrated that KEA3 contributes to efficient photosynthesis under fluctuating light conditions. The disturbed proton gradient regulation is a dominant mutant allele of KEA3, and its mutant phenotype is evident after a long period of dark adaptation (overnight; Wang et al., 2017). KEA3 is likely important during the induction of photosynthesis as well as under fluctuating light intensities. The similarity between the two conditions suggests that KEA3 is required for readjusting the ∆pH-dependent regulation immediately after any drastic change in light conditions.In this study, we characterized double mutants defective in the CET pathways and KEA3 to understand whether and how the synergy between CET and KEA3 in the regulatory network of photosynthesis affects this process. We focused on the contribution of NDH-dependent CET during the induction of photosynthesis after overnight dark adaptation in the kea3-1 mutant context. Based on our results, we propose a novel physiological function of the NDH complex: that of allowing flexibility of the regulatory network during the induction of photosynthesis.     

A Self-Excising Cre Recombinase Allows Efficient Recombination of Multiple Ectopic Heterospecific Lox Sites in Transgenic Tobacco

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, cre ^INT , was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising cre ^INT , sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with cre ^INT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The cre ^INT system has therefore advantages over systems with a continuously present Cre. The cre ^INT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants.     

Segmentation of the Vertebrate Head

Historical views of head segmentation are reviewed. The concensusis that the head is segmented essentially in terms of myomeres,and that other organs have responded in varying degrees to this.From the various lines of reasoning a model of the primitivevertebrate is generated. This model denies the tunicate originof the vertebrates—rather it identifies amphioxus as mostlike the ancestral vertebrate. The vertebrate head is made upof a preoral segment plus four other segments. Because of sclerotomites,the head extends through five and a half segments. The nasalorgans and eyes are preoral structures while the ear is locatedbetween segments three and four. The occipital portion of thehead skeleton is formed from the posterior half of the fifthsegment and the anterior half of the sixth; it is vertebra-likein structure. This "segment" is much altered as a result ofthe multiplication of the visceral pouches and is often viewedas the fusion product of several segments. Thus the idea ofcorrespondence between somite and visceral segments posteriorto the second branchial arch is rejected. In some fishes, additionalvertebrae are added to the posterior part of the cranium andthis can be observed in development. The bony cranium of thevertebrate appears to partially reflect segmentation; its componentssuggest a vertebra-like developmental influence in operation.Study of the shark head has contributed much to our knowledgeof this area.     

Novel Neuropeptide Y Y2-Like Receptor Subtype in Zebrafish and Frogs Supports Early Vertebrate Chromosome Duplications

The Y receptors comprise a family of G-protein coupled receptors with neuropeptide Y-family peptides as endogenous ligands. The Y receptor family has five members in mammals and evolutionary data suggest that it diversified in the two genome duplications proposed to have occurred early in vertebrate evolution. If this theory holds true, it allows for additional family members to be present. We describe here the cloning, pharmacological characterization, tissue distribution, and chromosomal localization of a novel subtype of the Y-receptor family, named Y7, from the zebrafish. We also present Y7 sequences from rainbow trout and two amphibians. The new receptor is most similar to Y2, with 51–54% identity. As Y2 has also been cloned from some of these species, there clearly are two separate Y2-subfamily genes. Chromosomal mapping in zebrafish supports origin of Y7 as a duplicate of Y2 by chromosome duplication in an early vertebrate. Y7 has probably been lost in the lineage leading to mammals. The pharmacological profile of the zebrafish Y7 receptor is different from mammalian Y2, as it does not bind short fragments of NPY with a high affinity. The Y7 receptor supports the theory of early vertebrate genome duplications and suggests that the Y family of receptors is a result of these early genome duplications.     

An Efficient Method for the Isolation and Purification of Oligoribonucleotides

Abstract

Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.