bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

A new gypsy-type retrotransposon, RIRE7: preferential insertion into the tandem repeat sequence TrsD in pericentromeric heterochromatin regions of rice chromosomes

A portion of an insertion sequence present in a member of the RIRE3 family of retrotransposons in Oryza sativa L. cv. IR36 was found to have an LTR sequence followed by a PBS sequence complementary to the 3'-end region of tRNAMet, indicative of another rice retrotransposon (named RIRE7). Cloning and sequencing of PCR-amplified fragments that made up all parts of the RIRE7 sequence showed that RIRE7 is a gypsy-type retrotransposon with partial homology in the pol region to the rice gypsy-type retrotransposons RIRE2 and RIRE3 identified in rice previously. Interestingly, various portions of the RIRE7 sequence were homologous to several DNA segments present in the centromere regions of cereal chromosomes. Further cloning and nucleotide sequencing of fragments flanking RIRE7 copies showed that RIRE7 was inserted into a site within a tandem repeat sequence that has a unit length of 155 bp. The tandem repeat sequence, named TrsD, was homologous to tandem repeat sequences RCS2 and CentC, previously identified in the centromeric regions of rice and maize chromosomes. Fluorescence in situ hybridization (FISH) analysis of the metaphase chromosomes of O. sativa cv. Nipponbare showed that both RIRE7 and TrsD sequences were present in the centromere regions of the chromosomes. The presence of RIRE7 and the TrsD sequences in the centromere regions of several chromosomes was confirmed by the identification of several YAC clones whose chromosomal locations are known. Further FISH analysis of rice pachytene chromosomes showed that the TrsD sequences were located in a pericentromeric heterochromatin region. These findings strongly suggest that RIRE7 and TrsD are components of the pericentromeric heterochromatin of rice chromosomes.     

A novel major quantitative trait locus controlling seed development at low temperature in soybean (Glycine max)

Low temperature is among the critical environmental factors that limit soybean production. To elucidate the genetic basis for chilling tolerance and identify useful markers, we conducted quantitative trait loci (QTL) analysis of seed-yielding ability at low temperature in soybean (Glycine max), using artificial climatic environments at usual and low temperatures and recombinant inbred lines derived from a cross between two contrasting cultivars in terms of chilling tolerance. We identified a QTL of a large effect (LOD > 15, r ² > 0.3) associated with seed-yielding ability only at low temperature. The QTL was mapped near marker Sat_162 on linkage group A2, where no QTL for chilling tolerance has previously been identified. The tolerant genotype did not increase the pod number but maintained the seed number per pod and single seed weight, namely, the efficiency of seed development at low temperature. The effect of the QTL was confirmed in a segregating population of heterogeneous inbred families, which provided near-isogenic lines. The genomic region containing the QTL also influenced the node and pod numbers regardless of temperature condition, although this effect was not primarily associated with chilling tolerance. These results suggest the presence of a new major genetic factor that controls seed development specifically at low temperature. The findings will be useful for marker-assisted selection as well as for understanding of the mechanism underlying chilling tolerance in reproductive organs.     

A long terminal repeat retrotransposon of fission yeast has strong preferences for specific sites of insertion

The successful dispersal of transposons depends on the critical balance between the fitness of the host and the ability of the transposon to insert into the host genome. One method transposons may use to avoid the disruption of coding sequences is to target integration into safe havens. We explored the interaction between the long terminal repeat retrotransposon Tf1 and the genome of the yeast Schizosaccharomyces pombe. Using techniques that were specifically designed to detect integration of Tf1 throughout the genome and to avoid bias in this detection, we generated 51 insertion events. Although 60.2% of the genome of S. pombe is coding sequence, all but one of the insertions occurred in intergenic regions. We also found that Tf1 was significantly more likely to insert into intergenic regions that included polymerase II promoters than into regions between convergent gene pairs. Interestingly, 8 of the 51 insertion sites were isolated multiple times from genetically independent cultures. This result suggests that specific sites in intergenic regions are targeted by Tf1. Perhaps the most surprising observation was that per kilobase of nonrepetitive sequence, Tf1 was significantly more likely to insert into chromosome 3 than into one of the other two chromosomes. This preference was found not to be due to differences in the distribution or composition of intergenic sequences within the three chromosomes.     

A transgenic locus in soybean exhibits a high level of recombination

Summary We have examined transgene inheritance in over 300 progeny of a line of soybean (Glycine max) transformed by particle bombardment with a construct containing bovine β-casein under the control of the soybean lectin 5′ and 3′ regulatory elements. Four copies of the casein transgene, located at a single locus, exhibit a high frequency of recombination that resulted in novel patterns in approximately 16% of the progeny in both the T₁ and T₂ generations. Characterization of the transgene locus using restriction enzymes that do not cut the transformation plasmid showed that all four transgene copies are at a single locus no larger than approximately 40 kb in size. Therefore, the recombination events resulting in the loss of transgene DNA are taking place within a limited physical distance on the host chromosome. This is the first report extensively documenting transgene instability at the DNA level in a plant transformed via particle bombardment. As this report indicates, a seemingly simple phenotype (presence of the foreign protein) may conceal inherent genetic instability at the DNA level.     

A single nuclear locus phylogeny of soybean based on DNA sequence

Soybean [Glycine max (L.) Merr.] evolution was examined by sequencing portions of the restriction fragment length polymorphism (RFLP) locus A-199a of 21 taxa from the Glycininae and 1 from the Phaseoleae. Four hundred nucleotides were determined in each, aligned, and then compared for these taxa. Within the annual soybean subgenus (Soja), the four accessions differed at as many as 2.2% of the nucleotides. Among 13 perennial soybean species (subgenus Glycine), nucleotide variation ranged from 1.7% to 8.4%. The nucleotide difference between the two soybean subgenera was 3.0–7.0%. Nucleotide variation between the genus Glycine and the related genera of Neonotonia, Amphicarpa, Teramnus, and Phaseolus ranged from 8.2% to 16.4%. In addition to nucleotide substitutions, insertions/deletions (indels) differences were also observed and were consistent with nucleotide-based analysis. Cladistic analysis of the A-199a sequences was performed using Wagner parsimony to construct a soybean phylogeny. Sixteen equally parsimonious trees were produced from these data. The trees were 246 steps in length with a consistency index of 0.78. Indels distribution upon the consensus topology revealed a pattern congruent with the nucleotide-based phylogeny. The current taxonomic status of the soybean subgenera and the related genera of Neonotonia, Amphicarpa, and Teramnus were well-supported and appear monophyletic in this analysis. Homoplasy within the subgenus Glycine led to a lack of resolved topology for many of these 13 taxa. However, the Glycine clade topology was consistent with phylogenies proposed using crossing experiments and cpDNA RFLPs. These genera were arranged from ancestral to derived as: Teramnus, Amphicarpa, Neonotonia, and Glycine when Phaseolus vulgaris was used as an outgroup.     

Overcoming the genetic compensation response of soybean florigens to improve adaptation and yield at low latitudes

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Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.     

Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet

C57BL/6J mice were fed a high-fat, carbohydrate-free diet (HFD) for 9 mo. Approximately 50% of the mice became obese and diabetic (ObD), approximately 10% lean and diabetic (LD), approximately 10% lean and nondiabetic (LnD), and approximately 30% displayed intermediate phenotype. All of the HFD mice were insulin resistant. In the fasted state, whole body glucose clearance was reduced in ObD mice, unchanged in the LD mice, and increased in the LnD mice compared with the normal-chow mice. Because fasted ObD mice were hyperinsulinemic and the lean mice slightly insulinopenic, there was no correlation between insulin levels and increased glucose utilization. In vivo, tissue glucose uptake assessed by 2-[(14)C]deoxyglucose accumulation was reduced in most muscles in the ObD mice but increased in the LnD mice compared with the values of the control mice. In the LD mice, the glucose uptake rates were reduced in extensor digitorum longus (EDL) and total hindlimb but increased in soleus, diaphragm, and heart. When assessed in vitro, glucose utilization rates in the absence and presence of insulin were similar in diaphragm, soleus, and EDL muscles isolated from all groups of mice. Thus, in genetically homogenous mice, HFD feeding lead to different metabolic adaptations. Whereas all of the mice became insulin resistant, this was associated, in obese mice, with decreased glucose clearance and hyperinsulinemia and, in lean mice, with increased glucose clearance in the presence of mild insulinopenia. Therefore, increased glucose clearance in lean mice could not be explained by increased insulin level, indicating that other in vivo mechanisms are triggered to control muscle glucose utilization. These adaptive mechanisms could participate in the protection against development of obesity.     

Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5'' coding region of the adenylate cyclase gene.

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.     

A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures.

The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy. Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da. Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E. coli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis, E. coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T. scotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P. haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.